Fusion pore formation and expansion induced by Ca2+ and synaptotagmin 1.

Fusion pore formation and expansion, crucial steps for neurotransmitter release and vesicle recycling in soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE)-dependent vesicle fusion, have not been well studied in vitro due to the lack of a reliable content-mixing fusion assay. Using methods detecting the intervesicular mixing of small and large cargoes at a single-vesicle level, we found that the neuronal SNARE complexes have the capacity to drive membrane hemifusion. However, efficient fusion pore formation and expansion require synaptotagmin 1 and Ca(2+). Real-time measurements show that pore expansion detected by content mixing of large DNA cargoes occurs much slower than initial pore formation that transmits small cargoes. Slow pore expansion perhaps provides a time window for vesicles to escape the full collapse fusion pathway via alternative mechanisms such as kiss-and-run. The results also show that complexin 1 stimulates pore expansion significantly, which could put bias between two pathways of vesicle recycling.

Characterization of breast cancers with PI3K mutations in an academic practice setting using SNaPshot profiling.

Mutations in the PIK3CA gene are common in breast cancer and represent a clinically useful therapeutic target. Several larger, population-based studies have shown a positive prognostic significance associated with these mutations. This study aims to further identify characteristics of patients harboring PIK3CA mutations while evaluating the clinical impact of genomic testing for these mutations. Tumors from 312 patients at Vanderbilt-Ingram Cancer Center were analyzed for PIK3CA mutations using a multiplex screening assay (SNaPshot). Mutation rates, receptor status, histopathologic characteristics, and time to recurrence were assessed. The number of patients participating in clinical trials, specifically trials relating to the PIK3CA mutation, was examined. Statistically significant differences between wild-type and mutated tumors were determined using the Wilcoxon, Pearson, and Fischer exact tests. The PIK3CA mutation was found in 25 % of tumors tested. Patients with PIK3CA mutations were significantly more likely to express hormone receptors, be of lower combined histological grade, and have a reduced time to recurrence. Patients found to have a PIK3CA mutation were significantly more likely to enter a PIK3CA-specific clinical trial. In addition to confirming previously established positive prognostic characteristics of tumors harboring PIK3CA mutations, this study demonstrates the feasibility and utility of mutation profiling in a clinical setting. PIK3CA mutation testing impacted treatment and resulted in more patients entering mutation-specific clinical trials.

A bioinformatics workflow for variant peptide detection in shotgun proteomics.

Shotgun proteomics data analysis usually relies on database search. However, commonly used protein sequence databases do not contain information on protein variants and thus prevent variant peptides and proteins from been identified. Including known coding variations into protein sequence databases could help alleviate this problem. Based on our recently published human Cancer Proteome Variation Database, we have created a protein sequence database that comprehensively annotates thousands of cancer-related coding variants collected in the Cancer Proteome Variation Database as well as noncancer-specific ones from the Single Nucleotide Polymorphism Database (dbSNP). Using this database, we then developed a data analysis workflow for variant peptide identification in shotgun proteomics. The high risk of false positive variant identifications was addressed by a modified false discovery rate estimation method. Analysis of colorectal cancer cell lines SW480, RKO, and HCT-116 revealed a total of 81 peptides that contain either noncancer-specific or cancer-related variations. Twenty-three out of 26 variants randomly selected from the 81 were confirmed by genomic sequencing. We further applied the workflow on data sets from three individual colorectal tumor specimens. A total of 204 distinct variant peptides were detected, and five carried known cancer-related mutations. Each individual showed a specific pattern of cancer-related mutations, suggesting potential use of this type of information for personalized medicine. Compatibility of the workflow has been tested with four popular database search engines including Sequest, Mascot, X!Tandem, and MyriMatch. In summary, we have developed a workflow that effectively uses existing genomic data to enable variant peptide detection in proteomics.

Genetically informed lung cancer medicine.

Knowledge of tumour mutation status has become increasingly important for the prioritization of targeted therapies for cancer. We review here the major known 'driver' mutations with therapeutic relevance in non-small cell lung cancer. We also discuss a variety of methods now being employed in molecular diagnostic laboratories to detect genetic alterations in lung tumours. A genetically-informed approach to lung cancer medicine is rapidly becoming the standard of care worldwide and should lead to improved outcomes for patients.

Hemifusion in SNARE-mediated membrane fusion.

SNAREs are essential for intracellular membrane fusion. Using EPR, we determined the structure of the transmembrane domain (TMD) of the vesicle (v)-SNARE Snc2p involved in trafficking in yeast. Structural features of the TMD were used to design a v-SNARE mutant in which about half of the TMD was deleted. Liposomes containing this mutant induced outer leaflet mixing but not inner leaflet mixing when incubated with liposomes containing target membrane (t)-SNAREs. Hemifusion was also detected with wild-type SNAREs when low protein concentrations were reconstituted. Thus, these results show that SNARE-mediated fusion can transit through a hemifusion intermediate.

The SNARE complex from yeast is partially unstructured on the membrane.

Molecular recognition between cognate SNAREs leads to the formation of a four-helix bundle, which facilitates vesicle docking and membrane fusion. For a SNARE system involved in trafficking in yeast, target membrane (t-) SNARE Sso1p and vesicle associated (v-) SNARE Snc2p contribute one SNARE motif each, whereas another t-SNARE (Sec9) donates two N-terminal and C-terminal SNARE motifs (SN1 and SN2) to the helical bundle. By use of EPR, it is found that SN2 has a tendency to be uncoiled, leaving a significant population of the SNARE complexes to be partially unstructured on the membrane. In sharp contrast, SN2 is fully engaged in the four-helix bundle when removed from the membrane, showing that the membrane is the main destabilizing factor. Helix-breaking proline mutations in SN2 did not affect the rate of docking but reduced the rate of lipid mixing significantly, indicating that SN2 plays an essential role in activating the transition from docking to fusion.

Fusion step-specific influence of cholesterol on SNARE-mediated membrane fusion.

Cholesterol is a major component of biological membranes and is known to affect vesicle fusion. However, the mechanism by which cholesterol modulates SNARE-dependent intracellular fusion is not well understood. Using the fluorescence assay and dye-labeled SNAREs and the fluorescent lipids, we dissected cholesterol effects on individual fusion steps including SNARE complex formation, hemifusion, pore formation, and pore dilation. At physiological high concentrations, cholesterol stimulated hemifusion as much as 30-fold, but its stimulatory effect diminished to 10-fold and three-fold for subsequent pore formation and pore expansion at 40 mol %, respectively. The results show that cholesterol serves as a strong stimulator for hemifusion but acts as mild stimulators for pore opening and expansion. Strong stimulation of hemifusion and mild stimulation of pore formation are consistent with the fusion model based on the intrinsic negative curvature of cholesterol. However, even a milder effect of cholesterol on pore expansion is contradictory to such a simple curvature-based prediction. Thus, we speculate that cholesterol also affects the conformation of the transmembrane domains of SNAREs, which modulates the fusion kinetics.

Functional KRAS mutations and a potential role for PI3K/AKT activation in Wilms tumors.

Wilms tumor (WT) is the most common renal neoplasm of childhood and affects 1 in 10 000 children aged less than 15 years. These embryonal tumors are thought to arise from primitive nephrogenic rests that derive from the metanephric mesenchyme during kidney development and are characterized partly by increased Wnt/ß-catenin signaling. We previously showed that coordinate activation of Ras and ß-catenin accelerates the growth and metastatic progression of a murine WT model. Here, we show that activating KRAS mutations can be found in human WT. In addition, high levels of phosphorylated AKT are present in the majority of WT. We further show in a mouse model and in renal epithelial cells that Ras cooperates with ß-catenin to drive metastatic disease progression and promotes in vitro tumor cell growth, migration, and colony formation in soft agar. Cellular transformation and metastatic disease progression of WT cells are in part dependent on PI3K/AKT activation and are inhibited via pharmacological inhibition of this pathway. Our studies suggest both KRAS mutations and AKT activation are present in WT and may represent novel therapeutic targets for this disease.

25-Hydroxyvitamin D: analysis and clinical application.

25-Hydroxyvitamin D (25-OHD) is one of the most popular tests requested by clinicians nowadays because in addition to bone diseases, many non-skeletal disorders have been suggested to be linked to vitamin D deficiency or insufficiency. Methodologies used in clinical laboratories include competitive vitamin D protein binding assays (CPBA), immunoassays, high performance liquid chromatography (HPLC), and liquid chromatography-tandem mass spectrometry (LC-MS/MS). In this review article, we introduce the basic metabolism and physiology of vitamin D, key issues in the methods for 25-OHD measurement currently used in most clinical laboratories, and clinical applications of 25-OHD testing. We conclude that although the methodologies for 25-OHD testing have improved significantly, considerable bias between different methods and laboratories still exists. Therefore, standardization of the method is critical. The optimal 25-OHD levels should be determined based on the standardized method. Also, more studies are needed to further determine the relationship between vitamin D deficiency or insufficiency and non-skeletal diseases as well as daily vitamin D dose requirement for reducing the risk of non-skeletal diseases.

Detection and monitoring of ovarian cancer.

Ovarian cancer is the fifth leading cause of death among women in the United States and remains the most common cause of death from a gynecologic malignancy. Most ovarian cancers are diagnosed at an advanced stage in which 5-year survival rate is approximately 30%. Given that the 5-year survival rate is greater than 90% for women diagnosed at an early stage, early detection in women presenting with vague symptoms is crucial to improve outcome. Diagnosis of ovarian cancer is largely based on symptoms, imaging, and laboratory biomarkers. Overall, more than 200 potential biomarkers differentially expressed in ovarian cancer have been identified (Lokshin, 2012). However, no single marker has been found useful for the diagnosis of ovarian cancer. Increased sensitivity and specificity for the diagnosis of ovarian cancer are observed when multiple markers are used in combination. The Food and Drug Administration (FDA) recently cleared two algorithms to evaluate the risk of ovarian cancer for women who present with pelvic mass. In this review, we will summarize the most recent serum biomarkers and clinical applications of biomarkers for the early detection and treatment monitoring of ovarian cancers. We will also discuss the algorithms for predicting the risk of ovarian cancers.

The recognition of 25-hydroxyvitamin D2 and D3 by a new binding protein based 25-hydroxyvitamin D assay.

BACKGROUND: The circulating form of vitamin D, 25-Hydroxyvitamin D (25-OHD), is commonly measured in clinical labs to evaluate vitamin D status of an individual. Vitamin D exists in 2 major forms: vitamin D3 and D2. Current methodologies for 25-OHD testing include antibody or vitamin D binding protein based assays, HPLC, and LC-MS. Although most current antibody or vitamin D binding protein based assays claim that they measure 25-OHD3 and 25-OHD2 equally, bias may still exist in the measurement of total 25-OHD when high concentration of 25-OHD2 is present. LC-MS/MS can measure 25-OHD3 and 25-OHD2 separately. We determined the ability of a new competitive protein binding assay (CPBA) to recognize 25-OHD2 and 25-OHD3 by comparing it with an LC-MS/MS method. METHODS: Seventy-one serum samples were collected and divided into 3 groups according to 25-OHD2 and 25-OHD3 concentrations determined by LC-MS/MS: group A containing only 25-OHD3 (25-OHD2<3ng/ml), group B containing both 25-OHD3 and 25-OHD2 (25-OHD2≥3ng/ml, 25-OHD3≥10ng/ml), and group C containing mainly 25-OHD2 (25-OHD3<10ng/ml). The correlation between total 25-OHD concentrations measured by LC-MS/MS and CPBA was analyzed with linear regression analysis and the difference between the two methods was analyzed with paired, 2-tailed Student's t-Test. The biases between the two methods were also calculated. RESULTS: The correlation analysis between LC-MS/MS (x) and CPBA (y) in the 3 groups are as follows: y=1.13x-1.01 (R(2)=0.65, n=25) in group A; y=0.68x+4.02 (R(2)=0.76, n=31) in group B, and y=0.49x+6.53 (R(2)=0.38, n=15) in group C. In group A, the mean total 25-OHD measured with CPBA was higher than that with LC-MS/MS, while in groups B, CPBA results were lower, but the differences were not statistically significant (p=0.476 for group A and 0.059 for group B). In group C, the mean 25-OHD measured with CPBA was lower than that with LC-MS/MS and the difference was statistically significant (p=0.002). The average biases in total 25-OHD concentrations between LC-MS/MS and CPBA were 10.8%, -23.6%, and -38.4% for groups A, B, and C, respectively. CONCLUSIONS: CPBA has a better correlation with LC-MS/MS for samples containing both 25-OHD2 and 25-OHD3 as compared to samples with mainly 25-OHD2 or 25-OHD3 only. However, it measures 25-OHD3 more accurately than 25-OHD2. Compared to LC-MS/MS, CPBA overestimates 25-OHD3, but underestimates 25-OHD2.

A single vesicle-vesicle fusion assay for in vitro studies of SNAREs and accessory proteins.

SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins are a highly regulated class of membrane proteins that drive the efficient merger of two distinct lipid bilayers into one interconnected structure. This protocol describes our fluorescence resonance energy transfer (FRET)-based single vesicle-vesicle fusion assays for SNAREs and accessory proteins. Both lipid-mixing (with FRET pairs acting as lipophilic dyes in the membranes) and content-mixing assays (with FRET pairs present on a DNA hairpin that becomes linear via hybridization to a complementary DNA) are described. These assays can be used to detect substages such as docking, hemifusion, and pore expansion and full fusion. The details of flow cell preparation, protein-reconstituted vesicle preparation, data acquisition and analysis are described. These assays can be used to study the roles of various SNARE proteins, accessory proteins and effects of different lipid compositions on specific fusion steps. The total time required to finish one round of this protocol is 3­6 d.

Routine multiplex mutational profiling of melanomas enables enrollment in genotype-driven therapeutic trials.

PURPOSE: Knowledge of tumor mutation status is becoming increasingly important for the treatment of cancer, as mutation-specific inhibitors are being developed for clinical use that target only sub-populations of patients with particular tumor genotypes. Melanoma provides a recent example of this paradigm. We report here development, validation, and implementation of an assay designed to simultaneously detect 43 common somatic point mutations in 6 genes (BRAF, NRAS, KIT, GNAQ, GNA11, and CTNNB1) potentially relevant to existing and emerging targeted therapies specifically in melanoma. METHODS: The test utilizes the SNaPshot method (multiplex PCR, multiplex primer extension, and capillary electrophoresis) and can be performed rapidly with high sensitivity (requiring 5-10% mutant allele frequency) and minimal amounts of DNA (10-20 nanograms). The assay was validated using cell lines, fresh-frozen tissue, and formalin-fixed paraffin embedded tissue. Clinical characteristics and the impact on clinical trial enrollment were then assessed for the first 150 melanoma patients whose tumors were genotyped in the Vanderbilt molecular diagnostics lab. RESULTS: Directing this test to a single disease, 90 of 150 (60%) melanomas from sites throughout the body harbored a mutation tested, including 57, 23, 6, 3, and 2 mutations in BRAF, NRAS, GNAQ, KIT, and CTNNB1, respectively. Among BRAF V600 mutations, 79%, 12%, 5%, and 4% were V600E, V600K, V600R, and V600M, respectively. 23 of 54 (43%) patients with mutation harboring metastatic disease were subsequently enrolled in genotype-driven trials. CONCLUSION: We present development of a simple mutational profiling screen for clinically relevant mutations in melanoma. Adoption of this genetically-informed approach to the treatment of melanoma has already had an impact on clinical trial enrollment and prioritization of therapy for patients with the disease.

BRAF(L597) mutations in melanoma are associated with sensitivity to MEK inhibitors.

UNLABELLED: Kinase inhibitors are accepted treatment for metastatic melanomas that harbor specific driver mutations in BRAF or KIT, but only 40% to 50% of cases are positive. To uncover other potential targetable mutations, we conducted whole-genome sequencing of a highly aggressive BRAF (V600) and KIT (W557, V559, L576, K642, and D816) wild-type melanoma. Surprisingly, we found a somatic BRAF(L597R) mutation in exon 15. Analysis of BRAF exon 15 in 49 tumors negative for BRAF(V600) mutations as well as driver mutations in KIT, NRAS, GNAQ, and GNA11, showed that two (4%) harbored L597 mutations and another two involved BRAF D594 and K601 mutations. In vitro signaling induced by L597R/S/Q mutants was suppressed by mitogen-activated protein (MAP)/extracellular signal-regulated kinase (ERK) kinase (MEK) inhibition. A patient with BRAF(L597S) mutant metastatic melanoma responded significantly to treatment with the MEK inhibitor, TAK-733. Collectively, these data show clinical significance to BRAF(L597) mutations in melanoma. SIGNIFICANCE: This study shows that cells harboring BRAF(L597R) mutants are sensitive to MEK inhibitor treatment, providing a rationale for routine screening and therapy of BRAF(L597R)-mutant melanoma.

Epidermal growth factor receptor mutation-guided treatment for lung cancers: Where are we now?

Epidermal growth factor receptor (EGFR) kinase domain mutations (from exon 18 to 21) alter the efficacy of the EGFR tyrosine kinase inhibitors gefitinib (Iressa) and erlotinib (Tarceva). Lung cancer patients with drug-sensitive EGFR mutations initially respond, but acquire resistance after approximately one year of tyrosine kinase inhibitor treatment. This review summarizes EGFR gene mutation profiles for East Asian non-small-cell lung cancer patients according to recent publications. Strategies for the treatment of acquired resistance mediated by EGFR T790M are also reviewed.

A platform for rapid detection of multiple oncogenic mutations with relevance to targeted therapy in non-small-cell lung cancer.

The identification of somatically acquired tumor mutations is increasingly important in the clinical management of cancer because the sensitivity of targeted drugs is related to the genetic makeup of individual tumors. Thus, mutational profiles of tumors can help prioritize anticancer therapy. We report herein the development and validation of two multiplexed assays designed to detect in DNA from FFPE tissue more than 40 recurrent mutations in nine genes relevant to existing and emerging targeted therapies in lung cancer. The platform involves two methods: a screen (SNaPshot) based on multiplex PCR, primer extension, and capillary electrophoresis that was designed to assess for 38 somatic mutations in eight genes (AKT1, BRAF, EGFR, KRAS, MEK1, NRAS, PIK3CA, and PTEN) and a PCR-based sizing assay that assesses for EGFR exon 19 deletions, EGFR exon 20 insertions, and HER2 exon 20 insertions. Both the SNaPshot and sizing assays can be performed rapidly, with minimal amounts of genetic material. Compared with direct sequencing, in which mutant DNA needs to compose 25% or more of the total DNA to easily detect a mutation, the SNaPshot and sizing assays can detect mutations in samples in which mutant DNA composes 1.56% to 12.5% and 1.56% to 6.25% of the total DNA, respectively. These robust, reliable, and relatively inexpensive assays should help accelerate adoption of a genotype-driven approach in the treatment of lung cancer.

A single-vesicle content mixing assay for SNARE-mediated membrane fusion.

The in vitro studies of membrane fusion mediated by soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) have primarily been conducted by following the mixing of lipids. However, the formation of a fusion pore and its expansion has been difficult to detect directly because of the leakiness of proteoliposomes, vesicle aggregation and rupture that often complicate the interpretation of ensemble fusion experiments. Fusion pore expansion is an essential step for full-collapse fusion and for recycling of fusion mechanisms. Here, we demonstrate a method to detect the inter-vesicular mixing of large cargoes at the single-molecule and -vesicle level. The change in fluorescence resonance energy transfer signal when a DNA hairpin encapsulated in a surface-tethered vesicle encounters a complementary DNA strand from another vesicle indicates content mixing. We found that the yeast SNARE complex alone without any accessory proteins can expand the fusion pore large enough to transmit ~11 kDa cargoes.

Single-Vesicle Fusion Assay Reveals Munc18-1 Binding to the SNARE Core Is Sufficient for Stimulating Membrane Fusion.

Munc18, an essential regulatory protein for intracellular membrane fusion mediated by SNAREs, is known for stabilizing the closed conformation of syntaxin through the interaction with the N-terminal Habc domain (amino acids 28-146) of syntaxin. In addition, Munc18 accelerates membrane fusion and its interaction with SNARE core and the N-peptide (amino acids 1-24) of syntaxin is thought to be necessary for this function. Using the recently developed fluorescence resonance energy transfer assay to detect the fusion between two individual vesicles harboring cognate SNARE proteins, we studied the effect of Munc18 on the fusion induced by neuronal SNARE proteins by following the mixing of lipid molecules between the two vesicles. We found that Munc18-1 stimulates neuronal SNARE-mediated fusion not only with full-length syntaxin 1A but also with a truncated syntaxin 1A that is missing both the Habc domain and the N-peptide. The electron paramagnetic resonance analysis indicates that the SNARE core/Munc18 interaction is responsible for this stimulatory function and the membrane plays a role for establishing this interaction.