Analysis of microRNAs in pancreatic fine-needle aspirates can classify benign and malignant tissues.

BACKGROUND: MicroRNAs (miRNAs) are RNA molecules that are involved in the regulation of many cellular processes, including those related to human cancers. The aim of this study was to determine, as a proof of principle, whether specific candidate miRNAs could be detected in fine-needle aspirate (FNA) biopsies of pancreatic ductal adenocarcinoma (PDAC) and could accurately differentiate malignant from benign pancreatic tissues. METHODS: We used TaqMan(R) assays to quantify miRNA levels in FNA samples collected in RNARetain (n = 16) and compared the results with a training set consisting of frozen macrodissected pancreatic samples (n = 20). RESULTS: Quantitative reverse-transcription PCR analysis confirmed that miRNA levels are affected in PDAC FNAs and correlate well with the changes observed in the training set of frozen pancreatic samples. Analysis of the amounts produced for a few specific miRNAs enabled identification of PDAC samples. The combination of miR-196a and miR-217 biomarkers further improved the ability to distinguish between healthy tissue, PDAC, and chronic pancreatitis in the training set (P = 8.2 x 10(-10)), as well as segregate PDAC FNA samples from other FNA samples (P = 1.1 x 10(-5)). Furthermore, we showed that miR-196a production is likely specific to PDAC cells and that its incidence paralleled the progression of PDAC. CONCLUSIONS: To the best of our knowledge, this study is the first to evaluate the diagnostic potential of miRNAs in a clinical setting and has shown that miRNA analysis of pancreatic FNA biopsy samples can aid in the pathologic evaluation of suspicious cases and may provide a new strategy for improving the diagnosis of pancreatic diseases.

Accurate molecular characterization of formalin-fixed, paraffin-embedded tissues by microRNA expression profiling.

Formalin-fixed, paraffin-embedded tissues are an invaluable tool for biomarker discovery and validation. As these archived specimens are not always compatible with modern genomic techniques such as gene expression arrays, we assessed the use of microRNA (miRNA) as an alternative means for the reliable molecular characterization of formalin-fixed, paraffin-embedded tissues. Expression profiling using two different microarray platforms and multiple mouse and human formalin-fixed, paraffin-embedded tissue types resulted in the correlation ratios of miRNA expression levels between frozen and fixed tissue pairs ranging from 0.82 to 0.99, depending on the cellular heterogeneity of the tissue type. The same miRNAs were identified as differentially expressed between tissues using both fixed and frozen specimens. While formalin fixation time had only marginal effects on microarray performance, extended storage times for tissue blocks (up to 11 years) resulted in a gradual loss of detection of miRNAs expressed at low levels. Method reproducibility and accuracy were also evaluated in two different tissues stored for different lengths of time. The technical variation between full process replicates, including independent RNA isolation methods, was approximately 5%, and the correlation of expression levels between microarray and real-time quantitative reverse transcriptase polymerase chain reaction was 0.98. Together, these data demonstrate that miRNA expression profiling is an accurate and robust method for the molecular analysis of archived clinical specimens, potentially extending the use of miRNAs as new diagnostic, prognostic, and treatment response biomarkers.

Evaluation and validation of total RNA extraction methods for microRNA expression analyses in formalin-fixed, paraffin-embedded tissues.

Histopathology archives of well-annotated formalin-fixed, paraffin-embedded (FFPE) tissue specimens are valuable resources for retrospective studies of human diseases. Since recovery of quality intact mRNA compatible with molecular techniques is often difficult due to degradation, we evaluated microRNA (miRNA), a novel class of small RNA molecules with growing therapeutic and diagnostic potential, as an alternative analyte for gene expression studies of FFPE samples. Analyzing total RNA yield, miRNA recovery, and robustness of real-time polymerase chain reaction for miRNA, mRNA, and rRNA species, we compared the performance of commercially available RNA isolation kits and identified a preferred methodology. We further implemented modifications to increase tissue throughput and incorporate a quantitative Armored RNA process control to monitor RNA recovery efficiency. The optimized process was tested for reproducibility as well as interoperator and interday variability, and was validated with a set of 30 clinical samples. In addition, we demonstrated that, independent of FFPE block age and RNA quality, miRNAs generate quantitative reverse transcription-polymerase chain reaction signals that are more robust and better correlate with expression levels from frozen reference samples compared with longer mRNAs. Our broad study, including a total of 272 independent RNA isolations from 17 tissue types and 65 FFPE blocks up to 12 years old, indicates that miRNAs are not only suitable but are also likely superior analytes for the molecular characterization of compromised archived clinical specimens.

The malonyl transferase activity of type II polyketide synthase acyl carrier proteins.

Acyl carrier proteins (ACPs) play a fundamental role in directing intermediates among the enzyme active sites of fatty acid and polyketide synthases (PKSs). In this paper, we demonstrate that the Streptomyces coelicolor (S. coelicolor) actinorhodin (act) PKS ACP can catalyze transfer of malonate to type II S. coelicolor fatty acid synthase (FAS) and other PKS ACPs in vitro. The reciprocal transfer from S. coelicolor FAS ACP to a PKS ACP was not observed. Several mutations in both act ACP and S. coelicolor FAS ACP could be classified by their participation in either donation or acceptance of this malonyl group. These mutations indicated that self-malonylation and malonyl transfer could be completely decoupled, implying that they were separate processes and that a FAS ACP could be converted from a non-malonyl-transferring protein to one with malonyl transferase activity.

The colorectal microRNAome.

MicroRNAs (miRNAs) are a class of small noncoding RNAs that have important regulatory roles in multicellular organisms. The public miRNA database contains 321 human miRNA sequences, 234 of which have been experimentally verified. To explore the possibility that additional miRNAs are present in the human genome, we have developed an experimental approach called miRNA serial analysis of gene expression (miRAGE) and used it to perform the largest experimental analysis of human miRNAs to date. Sequence analysis of 273,966 small RNA tags from human colorectal cells allowed us to identify 200 known mature miRNAs, 133 novel miRNA candidates, and 112 previously uncharacterized miRNA* forms. To aid in the evaluation of candidate miRNAs, we disrupted the Dicer locus in three human colorectal cancer cell lines and examined known and novel miRNAs in these cells. These studies suggest that the human genome contains many more miRNAs than currently identified and provide an approach for the large-scale experimental cloning of novel human miRNAs in human tissues.

Optimization of aptamer microarray technology for multiple protein targets.

Aptamer-based microarrays for the quantitation of multiple protein analytes have been developed. A multiplex aptamer microarray was generated by printing two RNA aptamers (anti-lysozyme and anti-ricin) and two DNA aptamers (anti-IgE and anti-thrombin) on to either streptavidin (SA) or neutravidin (NA)-coated glass slides. However, substantial optimization was required in order to ensure the simultaneous function of the aptamer:analyte pairs. The effects of protein labeling, assay buffer, surface coating, and immobilization chemistry and orientation were investigated. A single buffer (PBS buffer containing 5 mM MgCl2 and 0.1% Tween 20) was found to work well with all the aptamers, even though this was not the buffer originally used in their selection, while neutravidin-coated slides yielded a lower detection limit, wider detection range, and more uniform background than streptavidin-coated slides. Incubation with Cy3-labeled proteins yielded sensitive, target-specific, and dose-dependent responses to each protein. Target protein concentrations as low as 72 pg/mL (5 pM, lysozyme), 15 ng/mL (0.5 nM, ricin), 1.9 ng/mL (0.01 nM, IgE), and 170 ng/mL (5 nM, thrombin) could be detected. These results show that aptamer arrays can potentially be used with numerous proteins in parallel, furthering the notion that aptamer arrays may be useful in proteomics.

Kinetic mechanism for p38 MAP kinase alpha. A partial rapid-equilibrium random-order ternary-complex mechanism for the phosphorylation of a protein substrate.

p38 Mitogen-activated protein kinase alpha (p38 MAPKalpha) is a member of the MAPK family. It is activated by cellular stresses and has a number of cellular substrates whose coordinated regulation mediates inflammatory responses. In addition, it is a useful anti-inflammatory drug target that has a high specificity for Ser-Pro or Thr-Pro motifs in proteins and contains a number of transcription factors as well as protein kinases in its catalog of known substrates. Fundamental to signal transduction research is the understanding of the kinetic mechanisms of protein kinases and other protein modifying enzymes. To achieve this end, because peptides often make only a subset of the full range of interactions made by proteins, protein substrates must be utilized to fully elucidate kinetic mechanisms. We show using an untagged highly active form of p38 MAPKalpha, expressed and purified from Escherichia coli[Szafranska AE, Luo X & Dalby KN (2005) Anal Biochem336, 1-10) that at pH 7.5, 10 mm Mg2+ and 27 degrees C p38 MAPKalpha phosphorylates ATF2Delta115 through a partial rapid-equilibrium random-order ternary-complex mechanism. This mechanism is supported by a combination of steady-state substrate and inhibition kinetics, as well as microcalorimetry and published structural studies. The steady-state kinetic experiments suggest that magnesium adenosine triphosphate (MgATP), adenylyl (beta,gamma-methylene) diphosphonic acid (MgAMP-PCP) and magnesium adenosine diphosphate (MgADP) bind p38 MAPKalpha with dissociation constants of KA = 360 microm, KI = 240 microm, and KI > 2000 microm, respectively. Calorimetry experiments suggest that MgAMP-PCP and MgADP bind the p38 MAPKalpha-ATF2Delta115 binary complex slightly more tightly than they do the free enzyme, with a dissociation constant of Kd approximately 70 microm. Interestingly, MgAMP-PCP exhibits a mixed inhibition pattern with respect to ATF2Delta115, whereas MgADP exhibits an uncompetitive-like pattern. This discrepancy occurs because MgADP, unlike MgAMP-PCP, binds the free enzyme weakly. Intriguingly, no inhibition by 2 mm adenine or 2 mm MgAMP was detected, suggesting that the presence of a beta-phosphate is essential for significant binding of an ATP analog to the enzyme. Surprisingly, we found that inhibition by the well-known p38 MAPKalpha inhibitor SB 203580 does not follow classical linear inhibition kinetics at concentrations > 100 nm, as previously suggested, demonstrating that caution must be used when interpreting kinetic experiments using this inhibitor.

Following in vitro activation of mitogen-activated protein kinases by mass spectrometry and tryptic peptide analysis: purifying fully activated p38 mitogen-activated protein kinase alpha.

p38 mitogen-activated protein kinase alpha (MAPKalpha) belongs to the MAPK subfamily, which plays a pivotal role in cell signal transduction, where it mediates responses to cell stresses and, to a lesser extent, growth factors. Although its cellular function has been under intense scrutiny since its initial discovery, little progress has been made in understanding its kinetic mechanism. A contributory factor has been the lack of a fast and rigorous method for the purification of activated p38 MAPKalpha in sufficient quantity and purity for biophysical studies. Here we present a method for the preparation of milligram quantities of activated p38 MAPKalpha, specifically phosphorylated on Thr180 and Tyr182. Purification of the inactive (unphosphorylated) p38 MAPKalpha is facilitated by an N-terminal hexahistidine tag. Removal of this tag from His6-p38 MAPKalpha, prior to its activation, is essential to ensure preparation of high yields of homogeneous, dually phosphorylated enzyme. Activation is achieved on incubation with a glutathione S-transferase (GST) fusion of the constitutively active mutant of the upstream activator, MKK6b (GST-MKK6b S207E T211E), in the presence of MgATP2-. Notably, we show that specific formation of activated p38 MAPKalpha can be quantified by following the formation of the bis-phosphorylated tryptic peptide, 173-HTDDEMT*GY*VATR-186, using [gamma-32P]adenosine triphosphate (ATP) as the phosphate source and reverse-phase high-performance liquid chromatography (HPLC) to separate the phosphopeptides. This approach offers the only means to specifically determine both stoichiometry and specificity of p38 MAPKalpha phosphorylation.

A kinetic approach towards understanding substrate interactions and the catalytic mechanism of the serine/threonine protein kinase ERK2: identifying a potential regulatory role for divalent magnesium.

We are interested in the mechanism and regulation of the extracellular regulated protein kinases, ERK1 and ERK2, due to their key roles in cellular signal transduction and disease. Both enzymes phosphorylate a large number of structurally disparate proteins upon activation by phorbol esters, serum and growth factors, and are activated through a protein kinase cascade, termed the mitogen activated protein kinase (MAPK) pathway. ERK2 catalyses the transfer of the gamma-phosphate of adenosine triphosphate to serine or threonine residues found in Ser-Pro or Thr-Pro motifs on proteins. Its catalytic mechanism is intriguing, because it appears to predominantly rely on interactions outside of the active site cleft to specify a substrate. To study ERK2, we developed a recombinant protein called EtsDelta138, which comprises residues 1-138 of the transcription factor Ets-1, an excellent substrate of ERK2. Here we review several steady-state kinetic experiments that reveal details of the ERK2 mechanism and a hitherto unknown process of ERK2 activation by free magnesium. The physiological relevance of this mechanism is discussed.

Two rate-limiting steps in the kinetic mechanism of the serine/threonine specific protein kinase ERK2: a case of fast phosphorylation followed by fast product release.

Extracellular regulated protein kinase 2 (ERK2) is a eukaryotic protein kinase whose activity is regulated by mitogenic stimuli. To gain insight into the catalytic properties of ERK2 and to complement structure-function studies, we undertook a pre-steady state kinetic analysis of the enzyme. To do this, ERK2 was quantitatively activated by MAPKK1 in vitro by monitoring the stoichiometry and site specificity of phosphorylation using a combination of protein mass spectrometry, tryptic peptide analysis, and (32)P radiolabeling. Using a quench-flow apparatus, MgATP(2-) was rapidly mixed (<1 ms) with both ERK2 and the protein substrate EtsDelta138 in the presence of a saturating total concentration (20 mM) of magnesium ion at 27 degrees C and pH 7.5. An exponential burst of product was observed over the first few milliseconds that followed mixing. This burst had an amplitude alpha of 0.44 and was followed by a slower linear phase. The pre-steady state burst is consistent with two partially rate-limiting enzymatic steps, which have the following rate constants: k(2) = 109 +/- 9 s(-1) and k(3) = 56 +/- 4 s(-1). These are attributed to rapid phosphorylation of EtsDelta138 and the process of product release, respectively. Single-turnover experiments provided an independent determination of k(2) (106 +/- 25 s(-1)). The observed catalytic constant (k(cat)(obs)) was found to be sensitive to the concentration of ERK2. The data fit a model in which ERK2 monomers form dimers and suggest that both the monomeric and dimeric forms of ERK2 are active with catalytic constants (k(cat)) of 25 and 37 s(-1), respectively. In addition, the model suggests that in the presence of saturating concentrations of both magnesium and substrates ERK2 subunits dissociate with a dissociation constant (K(d)) of 32 +/- 16 nM.

The type I rat fatty acid synthase ACP shows structural homology and analogous biochemical properties to type II ACPs.

While X-ray and NMR structures are now available for most components of the Type II fatty acid synthase (FAS), there are no structures for Type I FAS domains. A region from the mammalian (rat) FAS, including the putative acyl carrier protein (ACP), has been cloned and over-expressed. Here we report multinuclear, multidimensional NMR studies which show that this isolated ACP domain contains four alpha-helices (residues 8-16 [1]; 41-51 [2]; 58-63 [3] and 66-74 [4]) and an overall global fold characteristic of ACPs from both Type II FAS and polyketide synthases (PKSs). The overall length of the structured ACP domain (67 residues) is smaller than the structured regions of the Eschericia coli FAS ACP (75 residues), the actinorhodin PKS ACP (78 residues) and the Bacillus subtilis FAS ACP (76 residues). We further show that the rat FAS ACP is recognised as an efficient substrate by enzymes known to modify Type II ACPs including phosphopantetheinyl and malonyl transferases, but not by the heterologous S. coelicolor minimal polyketide synthase.

Kinetic and mechanistic analysis of the malonyl CoA:ACP transacylase from Streptomyces coelicolor indicates a single catalytically competent serine nucleophile at the active site.

The source of malonyl groups for polyketide and fatty acid biosynthesis is malonyl CoA. During fatty acid and polyketide biosynthesis, malonyl groups are normally transferred to the acyl carrier protein (ACP) component of the synthase by a malonyl CoA:holo-ACP transacylase (MCAT) enzyme. The fatty acid synthase (FAS) malonyl CoA:ACP transacylase from Streptomyces coelicolor was expressed in Escherichia coli as a hexahistidine-tagged (His(6)) fusion protein in high yield. The His(6)-MCAT was purified to homogeneity using standard techniques, and kinetic analysis of the malonylation of S. coelicolorFAS holo-ACP, catalyzed by His(6)-MCAT, gave K(infinity) (M) values of 73 (ACP) and 60 microM (malonyl CoA). A catalytic constant k (infinity) (M) of 450 s(-1) and specificity constants k (infinity) (M)/K (infinity) (M) of 6.2 (ACP) and 7.5 microM(-1) s(-1) (malonyl CoA) were measured. Malonyl transfer to the E. coli FAS holo-ACP, catalyzed by His(6)-MCAT, was less efficient (k (infinity) (M)/K (infinity) (M) was 10% of that of the S. coelicolor ACP). Incubation of MCAT with the serine specific agent PMSF caused inhibition of malonyl transfer to FAS ACPs, and an S97A MCAT mutant was incapable of catalyzing malonyl transfer. Our results show that in the reaction with FAS holo-ACPs the S. coelicolor MCAT is very similar to the E. coli MCAT paradigm in terms of its kinetic mechanism and active site residues. These results indicate that no other active site nucleophile is involved in catalysis as has been suggested to explain recently reported observations.