CIUSuite 3: Next-Generation CCS Calibration and Automated Data Analysis Tools for Gas-Phase Protein Unfolding Data.

Ion mobility-mass spectrometry (IM-MS) has become a technology deployed across a wide range of structural biology applications despite the challenges in characterizing closely related protein structures. Collision-induced unfolding (CIU) has emerged as a valuable technique for distinguishing closely related, iso-cross-sectional protein and protein complex ions through their distinct unfolding pathways in the gas phase. With the speed and sensitivity of CIU analyses, there has been a rapid growth of CIU-based assays, especially regarding biomolecular targets that remain challenging to assess and characterize with other structural biology tools. With information-rich CIU data, many software tools have been developed to automate laborious data analysis. However, with the recent development of new IM-MS technologies, such as cyclic IM-MS, CIU continues to evolve, necessitating improved data analysis tools to keep pace with new technologies and facilitating the automation of various data processing tasks. Here, we present CIUSuite 3, a software package that contains updated algorithms that support various IM-MS platforms and supports the automation of various data analysis tasks such as peak detection, multidimensional classification, and collision cross section (CCS) calibration. CIUSuite 3 uses local maxima searches along with peak width and prominence filters to detect peaks to automate CIU data extraction. To support both the primary CIU (CIU1) and secondary CIU (CIU2) experiments enabled by cyclic IM-MS, two-dimensional data preprocessing is deployed, which allows multidimensional classification. Our data suggest that additional dimensions in classification improve the overall accuracy of class assignments. CIUSuite 3 also supports CCS calibration for both traveling wave and drift tube IM-MS, and we demonstrate the accuracy of a new single-field CCS calibration method designed for drift tube IM-MS leveraging calibrant CIU data. Overall, CIUSuite 3 is positioned to support current and next-generation IM-MS and CIU assay development deployed in an automated format.

Site-specific chirality-conferred structural compaction differentially mediates the cytotoxicity of Aß42.

Growing evidence supports the confident association between distinct amyloid beta (Aß) isoforms and Alzheimer's Disease (AD) pathogenesis. As such, critical investigations seeking to uncover the translational factors contributing to Aß toxicity represent a venture of significant value. Herein, we comprehensively assess full-length Aß42 stereochemistry, with a specific focus on models that consider naturally-occurring isomerization of Asp and Ser residues. We customize various forms of d-isomerized Aß as natural mimics, ranging from fragments containing a single d residue to full length Aß42 that includes multiple isomerized residues, systematically evaluating their cytotoxicity against a neuronal cell line. Combining multidimensional ion mobility-mass spectrometry experimental data with replica exchange molecular dynamics simulations, we confirm that co-d-epimerization at Asp and Ser residues within Aß42 in both N-terminal and core regions effectively reduces its cytotoxicity. We provide evidence that this rescuing effect is associated with the differential and domain-specific compaction and remodeling of Aß42 secondary structure.

Comparing Selected-Ion Collision Induced Unfolding with All Ion Unfolding Methods for Comprehensive Protein Conformational Characterization.

Structural analysis by native ion mobility-mass spectrometry provides a direct means to characterize protein interactions, stability, and other biophysical properties of disease-associated biomolecules. Such information is often extracted from collision-induced unfolding (CIU) experiments, performed by ramping a voltage used to accelerate ions entering a trap cell prior to an ion mobility separator. Traditionally, to simplify data analysis and achieve confident ion identification, precursor ion selection with a quadrupole is performed prior to collisional activation. Only one charge state can be selected at one time, leading to an imbalance between the total time required to survey CIU data across all protein charge states and the resulting structural analysis efficiency. Furthermore, the arbitrary selection of a single charge state can inherently bias CIU analyses. We herein aim to compare two conformation sampling methods for protein gas-phase unfolding: (1) traditional quadrupole selection-based CIU and (2) nontargeted, charge selection-free and shotgun workflow, all ion unfolding (AIU). Additionally, we provide a new data interpretation method that integrates across all charge states to project collisional cross section (CCS) data acquired over a range of activation voltages to produce a single unfolding fingerprint, regardless of charge state distributions. We find that AIU in combination with CCS accumulation across all charges offers an opportunity to maximize protein conformational information with minimal time cost, where additional benefits include (1) an improved signal-to-noise ratios for unfolding fingerprints and (2) a higher tolerance to charge state shifts induced by either operating parameters or other factors that affect protein ionization efficiency.

Ion Mobility-Mass Spectrometry Reveals the Structures and Stabilities of Biotherapeutic Antibody Aggregates.

Stability is a key critical quality attribute monitored throughout the development of monoclonal antibody (mAb) therapeutics. Minor changes in their higher order structure (HOS) caused by stress or environment may alter mAb aggregation, immunogenicity, and efficacy. In addition, the structures of the resulting mAb aggregates are largely unknown, as are their dependencies on conditions under which they are created. In this report, we investigate the HOS of mAb monomers and dimers under a variety of forced degradation conditions with ion mobility-mass spectrometry (IM-MS) and collision-induced unfolding (CIU) technologies. We evaluate two model IgG1 antibodies that differ significantly only in their complementarity-determinant regions: IgG1α and IgG1ß. Our data covering both heat- and pH-based forced degradation conditions, aquired on two different IM-MS platforms, show that these mAbs undergo global HOS changes at both monomer and dimer levels upon degradation, but shifts in collision cross section (CCS) differ under pH or heat degradation conditions. In addition, the level of CCS change detected is different between IgG1α and IgG1ß, suggesting that differences in the CDR drive differential responses to degradation that influence the antibody HOS. Dramatically different CIU fingerprints are obtained for IgG1α and IgG1ß monomers and dimers for both degradation conditions. Finally, we constructed a series of computational models of mAb dimers for comparison with experimental CCS values and found evidence for a compact, overlapped dimer structure under native and heat degradation conditions, possibly adopting an inverted or nonoverlapped quaternary structure when produced through pH degredation. We conclude by discussing the potential impact of our findings on ongoing biotherapeutic discovery and development efforts.

Butyrate-mediated acquisition of chemoresistance by human colon cancer cells.

Butyrate is a short-chain fatty acid produced by the intestinal microflora and it not only induces apoptosis but also inhibits the proliferation of cancer cells. Recently, it has been reported that butyrate may cause resistance in colon cancer cells. Therefore, we investigated the effects of increased resistance to butyrate in HCT116 colon cancer cells. We established HCT116 cells resistant to butyrate (HCT116/BR) by treating HCT116 parental cells (HCT116/PT) with increasing concentrations of butyrate to a maximum of 1.6 mM for 3 months. The butyrate concentrations that inhibited cell growth by 50% (IC50) were 0.508 and 5.50 mM in HCT116/PT and HCT116/BR cells. The values after treatment with paclitaxel, 5-fluorouracil (5-FU), doxorubicin and trichostatin A (TSA) were 2.42, 2.36, 4.31 and 11.3-fold higher, respectively, in HCT116/BR cells compared with HCT116/PT cells. The protein expression of drug efflux pumps, such as P-glycoprotein (P-gp), breast cancer-resistant protein (BCRP) and the multidrug resistance associated protein 1 (MRP1), did not differ between HCT116/PT and HCT116/BR cells. The expression level of the anti-apoptotic Bcl-xL protein was increased while those of pro-apoptotic Bax and Bim proteins were reduced in HCT116/BR cells. There were no significant differences in cell motility and invasion. This study suggests that exposure of colon cancer cells to butyrate results in development of resistance to butyrate, which may play a role in the acquisition of chemoresistance in colon cancer.

Increased chemoresistance to paclitaxel in the MCF10AT series of human breast epithelial cancer cells.

The MCF10AT cell series of human breast epithelial cancer cells includes normal MCF10A (10A), premalignant MCF10AT (10AT) and MCF10ATG3B (10ATG3B), and fully malignant MCF10CA1a (10CA1a) cells. The series is a unique model system showing progressive tumorigenic potential with the same origin. The effects of paclitaxel, a microtubule inhibitor, were evaluated in this cell system. Paclitaxel inhibited cell proliferation in a time-dependent (24, 48 and 72 h) and concentration-dependent (0-10 nM) manners with less sensitivity in 10CA1a cells. Treatment with paclitaxel (10 nM) for 24 h induced apoptosis in 10A, 10AT, 10ATG3B and 10CA1a cells, with 23.6, 26.1, 25.2 and 8.96%, respectively, in the sub-G1 phase. Treatment with paclitaxel (0-10 nM) for 24 h, resulted in the appearance of DNA fragmentation (a hallmark of apoptosis) with less sensitivity in the 10CA1a tumor cells. Paclitaxel increased p53 protein expression in 10A, 10AT, 10ATG3B and 10CA1a cells, by 87, 102, 812 and 84%, respectively. The p21Waf1/Cip1 protein expression increased by 2.57-, 1.53- and 2.48-fold in 10A, 10AT and 10ATG3B cells, respectively, with negligible detection in the 10CA1a cells. Activation of the Akt signaling pathway was observed in the MCF10AT cell lineage and the protein expression of phospho-Akt (Ser473 and Thr308). The downstream targets of this pathway, phospho-p70S6K and phospho-S6RP, were also inhibited by paclitaxel in 10A, 10AT and 10ATG3B cells, but minimally inhibited in 10CA1a cells, suggestive of chemoresistance in 10CA1a cells. The effects of paclitaxel on the multidrug resistance 1 (MDR1), MRP1 and breast cancer resistance protein (BCRP) gene expression were not significant in the MCF10AT cell lineage. These results collectively indicated that paclitaxel inhibited cell proliferation and induced apoptosis in the MCF10AT cell lineage, with chemoresistance in 10CA1a tumor cells. The decreased responsiveness to paclitaxel observed in 10CA1a tumor cells was likely due, in part, to activation of the Akt signaling pathway and a high expression of wild-type p53 with lack of p21Waf1/Cip1.

Epigenetic regulation of vitamin D metabolism in human lung adenocarcinoma.

INTRODUCTION: 1α,25-Dihydroxyvitamin D3 (1,25-D3) is antiproliferative in preclinical models of lung cancer, but in tumor tissues, its efficacy may be limited by CYP24A1 expression. CYP24A1 is the rate limiting catabolic enzyme for 1,25-D3 and is overexpressed in human lung adenocarcinoma (AC) by unknown mechanisms. METHODS: The DNA methylation status of CYP24A1 was determined by bisulfite DNA pyrosequencing in a panel of 30 lung cell lines and 90 surgically resected lung AC. The level of CYP24A1 methylation was correlated with CYP24A1 expression in lung AC cell lines and tumors. In addition, histone modifications were assessed by quantitative chromatin immunoprecipitation-polymerase chain reaction (ChIP-qPCR) in A549, NCI-H460, and SK-LU-1. RESULTS: Bisulfite DNA pyrosequencing analysis revealed that CYP24A1 gene was heterogeneously methylated in lung AC. Expression of CYP24A1 was inversely correlated with promoter DNA methylation in lung AC cell lines and tumors. Treatment with 5-aza-2'-deoxycytidine (5-Aza) and trichostatin A (TSA) increased CYP24A1 expression in lung AC. We observed that CYP24A1 promoter hypermethylation decreased CYP24A1 enzyme activity in vitro, whereas treatment with 5-Aza and/or TSA increased CYP24A1 enzyme affinity for its substrate 1,25-D3. In addition, ChIP-qPCR analysis revealed specific histone modifications within the CYP24A1 promoter region. Treatment with TSA increased H3K4me2 and H3K9ac and simultaneously decreased H3K9me2 at the CYP24A1 promoter and treatment with 5-Aza and/or TSA increased the recruitment of vitamin D receptor (VDR) to vitamin D response elements (VDRE) of the CYP24A1 promoter. CONCLUSIONS: The expression of CYP24A1 gene in human lung AC is in part epigenetically regulated by promoter DNA methylation and repressive histone modifications. These findings should be taken into consideration when targeting CYP24A1 to optimize antiproliferative effects of 1,25-D3 in lung AC.

Characterization of vitamin D receptor (VDR) in lung adenocarcinoma.

PURPOSE: The anti-proliferative effects of 1α,25-dihydroxyvitamin D(3) (1,25-D(3), calcitriol, the active form of vitamin D) are mediated by the nuclear vitamin D receptor (VDR). In the present study, we characterized VDR expression in lung adenocarcinoma (AC). EXPERIMENTAL DESIGN: We examined VDR mRNA expression using a quantitative real-time PCR (qRT-PCR) in 100 patients who underwent surgery for lung AC. In a subset of these patients (n=89), we examined VDR protein expression using immunohistochemistry. We also examined the association of VDR protein expression with circulating serum levels of 25-hydroxyvitamin D(3) (25-D(3)) and 1,25-D(3). The antiproliferative effects and cell cycle arrest of 1,25-D(3) were examined using lung cancer cell lines with high (SKLU-1) as well as low (A549) expression of VDR mRNA. RESULTS: Higher VDR expression correlates with longer survival after adjusting for age, sex, disease stage and tumor grade (HR 0.73, 95% CI 0.58-0.91). In addition, there was a positive correlation (r=0.38) between serum 1,25-D(3) and tumor VDR protein expression. A greater anti-proliferative effect of 1,25-D(3) was observed in high compared to low VDR-expressing cell lines; these effects corresponded to G1 cell cycle arrest; this was associated with a decline in cyclin D1, S-phase kinase protein 2 (Skp2), retinoblastoma (Rb) and minichromosome maintenance 2 (MCM2) proteins involved in S-phase entry. CONCLUSIONS: Increased VDR expression in lung AC is associated with improved survival. This may relate to a lower proliferative status and G1 arrest in high VDR-expressing tumors.