HeLa Cell Extracts Research Articles

The Human Paraoxonase 2: An Optimized Procedure for Refolding and Stabilization Facilitates Enzyme Analyses and a Proteomics Approach.

The human paraoxonase 2 (PON2) is the oldest member of a small family of arylesterase and lactonase enzymes, representing the first line of defense against bacterial infections and having a major role in ROS-associated diseases such as cancer, cardiovascular diseases, neurodegeneration, and diabetes. Specific Post-Translational Modifications (PTMs) clustering nearby two residues corresponding to pon2 polymorphic sites and their impact on the catalytic activity are not yet fully understood. Thus, the goal of the present study was to develop an improved PON2 purification protocol to obtain a higher amount of protein suitable for in-depth biochemical studies and biotechnological applications. To this end, we also tested several compounds to stabilize the active monomeric form of the enzyme. Storing the enzyme at 4 °C with 30 mM Threalose had the best impact on the activity, which was preserved for at least 30 days. The catalytic parameters against the substrate 3-Oxo-dodecanoyl-Homoserine Lactone (3oxoC12-HSL) and the enzyme ability to interfere with the biofilm formation of Pseudomonas aeruginosa (PAO1) were determined, showing that the obtained enzyme is well suited for downstream applications. Finally, we used the purified rPON2 to detect, by the direct molecular fishing (DMF) method, new putative PON2 interactors from soluble extracts of HeLa cells.

Diagonal Chromatography for the Isolation of O-Linked β-N-acetyl-D-Glucosaminylated (O-GlcNAcylated) Peptides in HeLa Cell Extracts

Protein O-linked-N-acetylglucosaminylation (O-GlcNAcylation) is a dynamic post-translational modification process that plays an essential role in biological activities. Growing evidence indicates that aberration of O-GlcNAcylation is associated with various diseases, e.g. diabetes, neurological diseases, and cancers. However, the mechanistic studies of O-GlcNAcylation are lagging behind other post-translational modifications due to its extremely low abundance, limited analytical tools, and specificity. Herein, diagonal strong cation exchange chromatography was applied to enrich the O-GlcNAc glycosylated peptides prior to mass spectrometric analysis by liquid chromatography/ion trap tandem mass spectrometry (LC–MS/MS). In this strategy, the O-GlcNAcylated peptides were first enzymatically labeled with an azide-modified galactosamine (GalNAz) and fractionated by strong cation exchange (SCX) chromatography. Tris(carboxyethyl)phosphine (TCEP) reduces the azido group in GalNAz-modified peptides to a primary amine group. TCEP-induced reduction of GalNAz-modified peptides was separated from unmodified peptides by diagonal SCX. By reversed-phase LC–MS/MS analysis of secondary SCX fractions, O-GlcNAcylated peptides were isolated and identified from the mixtures of O-GlcNAc-modified and unmodified peptides in HeLa cell extract. A total of 250 O-GlcNAcylation sites on 215 proteins were identified. Therefore, this novel method could be a potential tool for the isolation and site analysis of O-GlcNAc-modified peptides.

Comprehensive Comparison of the Capacity of Functionalized Sepharose, Magnetic Core, and Polystyrene Nanoparticles to Immuno-Precipitate Procalcitonin from Human Material for the Subsequent Quantification by LC-MS/MS

Sepsis is a life-threatening organ dysfunction caused by a dysregulated host response to infection. The fast and accurate diagnosis of sepsis by procalcitonin (PCT) has emerged as an essential tool in clinical medicine. Although in use in the clinical laboratory for a long time, PCT quantification has not yet been standardized. The International Federation of Clinical Chemistry working group on the standardization of PCT (IFCC-WG PCT) aims to provide an LC-MS/MS-based reference method as well as the highest metrological order reference material to address this diagnostic need. Here, we present the systematic evaluation of the efficiency of an immuno-enrichment method, based on functionalized Sepharose, magnetic-core, or polystyrene (latex) nano-particles, to quantitatively precipitate PCT from different human sample materials. This method may be utilized for both mass spectrometric and proteomic purposes. In summary, only magnetic-core nano-particles functionalized by polyclonal PCT antibodies can fulfil the necessary requirements of the international standardization of PCT. An optimized method proved significant benefits in quantitative and specific precipitation as well as in the subsequent LC-MS/MS detection of PCT in human serum samples or HeLa cell extract. Based on this finding, further attempts of the PCT standardization process will utilize a magnetic core-derived immuno-enrichment step, combined with subsequent quantitative LC-MS/MS detection.

Multiple intra-articular injections with adipose-derived stem cells for knee osteoarthritis cause severe arthritis with anti-histone H2B antibody production

IntroductionOsteoarthritis (OA) is the most common form of arthritis. OA results from the breakdown of cartilage, which leads to deterioration of the entire joint and the connective tissue that holds the joint together, and gradually and irreversibly worsens over time. Adipose-derived stem/stromal cells (ADSCs) have been used in the treatment of knee OA. However, the safety and efficacy of ADSC treatment of OA remain unclear. In this study, we investigated the pathophysiology of severe knee arthritis that occurred after ADSC treatment by screening for autoantibodies in synovial fluid from patients who received ADSC treatment. MethodsAdult Japanese patients with OA who received ADSC treatment at Saitama Cooperative Hospital between June 2018 and October 2021 were enrolled. Antibodies (Abs) were screened using immunoprecipitation (IPP) with [35S]-methionine-labeled HeLa cell extracts. The detected protein was identified by liquid chromatography coupled with time-of-flight mass spectrometry (MS) and ion trap MS, and the corresponding proteins were confirmed as autoantigens using immunoblotting. Ab titers were measured using an enzyme-linked immunosorbent assay. ResultsA total of 113 patients received ADSC treatment, and 75% (85/113) received ADSC injection at least twice with a 6-month interval between. No obvious abnormalities were observed in any patient after their first treatment; by contrast, 53% (45/85) of patients who received their second or third ADSC injection showed severe knee arthritis. IPP detected a common anti-15 kDa Ab in synovial fluid of 62% (8/13) of the samples analyzed from patients who showed severe arthritis. This Ab was not detected in synovial fluid obtained from the same joints before treatment. The corresponding autoantigen was identified as histone H2B. All available synovial samples from patients who tested positive for anti-histone H2B Ab were newly positive after the treatment; that is, none had been positive for anti-histone H2B Ab before treatment. ConclusionsMultiple ADSC injections for OA induced severe arthritis in a high percentage of patients, particularly after the second injection. Synovial fluid from some patients with knee arthritis contained Ab to histone H2B that appeared only after ADSC treatment. These findings provide new insights into the pathogenesis of ADSC treatment-induced severe arthritis.

A nitrogen-doped graphene tube composite based on immobilized metal affinity chromatography for the capture of phosphopeptides

A novel immobilized metal affinity chromatography (IMAC) functional composite, mNi@N-GrT@PDA@Ti4+, was fabricated based on ultrathin magnetic nitrogen-doped graphene tube (mNi@N-GrT) after chelated Ti4+ with polydopamine, following as a magnetic solid-phase extraction sorbent for rapidly selective enrichment and mass spectrometry identification of phosphorylated peptides. After optimized, the composite exhibited high specificity in the enrichment of phosphopeptides from the digest mixture of β-casein and bovine serum albumin (BSA). The robust method presented the low detection limits (1 fmol, 200 μL) and excellent selectivity (1:100) in the molar ration mixture of β-casein and BSA digests. Furthermore, the selective enrichment of phosphopeptides in the complex bio-samples, was successfully carried out. The results showed that 28 phosphopeptides were finally detected in mouse brain, and 2087 phosphorylated peptides were identified in the HeLa cells extracts with specific selectivity of 95.6%. The enrichment performance of mNi@N-GrT@PDA@Ti4+ was satisfactory, suggesting that the functional composite provided a potential application in the enrichment of trace phosphorylated peptides from the complex biological matrix.

Pyridine Derivative of Succinic Acid Hydroxylamide Enhances the Cytotoxic Effect of Cisplatin and Actinomycin D.

We studied the possibility of inhibition of histone deacetylases (HDAC) in the nuclear extract of HeLa cells by N1-hydroxy-N4-(pyridin-4-yl)succinamide (compound 1). Compound 1 inhibits HDAC and showed low toxicity for A-172, HepG2, HeLa, MCF-7, and Vero cells. HeLa cells were most sensitive to the compound. Increasing the interval between administration of compound 1 and the chemotherapeutic agent to 8 h led to an increase in the cytotoxic effect of cisplatin (actinomycin D) on HeLa cells. The combination of compound 1 with cisplatin (actinomycin D) reduced the cytotoxic effect of these drugs for non-tumor Vero cells.

MiRNA accessibility in cellular RISC revealed by single molecule kinetic fingerprinting.

MicroRNAs (miRNAs) in association with Argonaute (Ago) proteins in the RNA induced silencing complex (RISC) regulate the levels of translation of messenger RNAs (mRNAs). The major parameter thought to explain the selection of the target mRNA is the base pairing between the ‘5’ seed’ region of the miRNA and its counterpart mRNA. Ago2 crystalizes in a conformation where the 5’ seed region is exposed to the target mRNA. However, both 5’ seed and 3’ non-seed-based target recognition has been reported. In fact, thousands of miRNA target sites depend on a non-seed pairing interaction, highlighting a gap in our understanding of miRNA targeting rules. Here, we present a ‘single molecule kinetic fingerprinting’ approach using split miRNA complementary probes (i.e., target mimics) that trace the differential accessibility of the 5’ seed and 3’ non-seed segments of the miRNA in RISC immunoprecipitated from human HeLa cell extract. Two distinct populations are observed with either the 5’ seed or the 3’ non-seed segment accessible. Only a minor population (∼5-10%) shows simultaneous accessibility of both segments. Compared to the protein-free miRNA, the target interaction kinetics of the seed and non-seed segments of the Ago2-bound miRNA are differentially controlled by the surrounding protein. In particular, tight target binding to the 5’ seed region alters the kinetics of the 3’ non-seed interaction, resulting in kinetic heterogeneity in that interaction with the target mimic. This kinetic heterogeneity most likely arises from differential accessibility of the 3’ non-seed region of miRNA in Ago2, which adopts multiple conformations. In summary, our data indicate that both 5’ seed and 3’ non-seed-based target recognition is possible, but the accessibility and the kinetics of mRNA interactions is largely governed by the surrounding protein conformation as well as the guide-target complementarity.

A New, Rapid Method for the Quantification of Dolichyl Phosphates in Cell Cultures Using TMSD Methylation Combined with LC-MS Analysis.

Dolichyl phosphates (DolP) are ubiquitous lipids that are present in almost all eukaryotic membranes. They play a key role in several protein glycosylation pathways and the formation of glycosylphosphatidylinositol anchors. These lipids constitute only ~0.1% of total phospholipids, and their analysis by reverse phase (RP) liquid chromatography-high-resolution mass spectrometry (LC-HRMS) is challenging due to their high lipophilicity (log P > 20), poor ionization efficiency, and relatively low abundance. To overcome these challenges, we have introduced a new approach for DolP analysis by combining trimethylsilyldiazomethane (TMSD)-based phosphate methylation and HRMS analysis. The analytical method was validated for its reproducibility, sensitivity, and accuracy. The established workflow was successfully applied for the simultaneous characterization and quantification of DolP species with different isoprene units in lipid extracts of HeLa and Saccharomyces cerevisiae cells.

Unified-Hydrophilic-Interaction/Anion-Exchange Liquid Chromatography Mass Spectrometry (Unified-HILIC/AEX/MS): A Single-Run Method for Comprehensive and Simultaneous Analysis of Polar Metabolome.

One of the technical challenges in the field of metabolomics is the development of a single-run method to detect the full complement of polar metabolites in biological samples. However, an ideal method to meet this demand has not yet been developed. Herein, we proposed a simple methodology that enables the comprehensive and simultaneous analysis of polar metabolites using unified-hydrophilic-interaction/anion-exchange liquid chromatography mass spectrometry (unified-HILIC/AEX/MS) with a polymer-based mixed amines column composed of methacrylate-based polymer particles with primary, secondary, tertiary, and quaternary amines as functional groups. The optimized unified-HILIC/AEX/MS method is composed of two consecutive chromatographic separations, HILIC-dominant separation for cationic, uncharged, and zwitterionic polar metabolites [retention times (RTs) = 0-12.8 min] and AEX-dominant separation for polar anionic metabolites (RTs = 12.8-26.5 min), by varying the ratio of acetonitrile to 40 mM ammonium bicarbonate solution (pH 9.8). A total of 400 polar metabolites were analyzed simultaneously through a combination of highly efficient separation using unified-HILIC/AEX and remarkably sensitive detection using multiple reaction monitoring-based triple quadrupole mass spectrometry (unified-HILIC/AEX/MS/MS). A nontargeted metabolomic approach using unified-HILIC/AEX high-resolution mass spectrometry (unified-HILIC/AEX/HRMS) also provided more comprehensive information on polar metabolites (3242 metabolic features) in HeLa cell extracts than the conventional HILIC/HRMS method (2068 metabolic features). Our established unified-HILIC/AEX/MS/MS and unified-HILIC/AEX/HRMS methods have several advantages over conventional techniques, including polar metabolome coverage, throughput, and accurate quantitative performance, and represent potentially useful tools for in-depth studies on metabolism and biomarker discovery.

Target recognition assisted-primer exchange reaction (Ta-PER) for sensitive analysis of p53 gene and its application in analyzing amatoxin-treated samples.

Sensitive and reliable detection of thep53 gene plays a significant role in precise cancer targeting and in fundamental research. However, thesensitivity of existing p53 gene detection approaches remains to be improved. Herein, we develop a target recognition assisted-primer exchange reaction (Ta-PER) for sensitive analysis of thep53 gene. Ta-PER was initiated by the recognition of a designed dumbbell structure probe by thep53 gene. In Ta-PER, theprimer exchange reaction (PER) was combined with molecular beacon-based chain recycling to construct the signal amplification process. Through integrating target recognition with PER-based signal amplification, Ta-PER was established and exhibited a high detection sensitivity, with alimit of detection as low as 56 fM. In addition, the approach was also used to detect thep53 gene in normal HeLa cells and amatoxin-treated HeLa cells. The high level of thep53 gene in amatoxin-treated HeLa cells, which was approximately 1.67 times higher than that in HeLa cell extract, indicated the apoptosis of cells and suggested the promising prospect of the approach.

Treatment of Human HeLa Cells with Black Raspberry Extracts Enhances the Removal of DNA Lesions by the Nucleotide Excision Repair Mechanism.

As demonstrated by us earlier and by other researchers, a diet containing freeze-dried black raspberries (BRB) inhibits DNA damage and carcinogenesis in animal models. We tested the hypothesis that the inhibition of DNA damage by BRB is due, in part, to the enhancement of DNA repair capacity evaluated in the human HeLa cell extract system, an established in vitro system for the assessment of cellular DNA repair activity. The pre-treatment of intact HeLa cells with BRB extracts (BRBE) enhances the nucleotide excision repair (NER) of a bulky deoxyguanosine adduct derived from the polycyclic aromatic carcinogen benzo[a]pyrene (BP-dG) by ~24%. The NER activity of an oxidatively-derived non-bulky DNA lesion, guanidinohydantoin (Gh), is also enhanced by ~24%, while its base excision repair activity is enhanced by only ~6%. Western Blot experiments indicate that the expression of selected, NER factors is also increased by BRBE treatment by ~73% (XPA), ~55% (XPB), while its effects on XPD was modest (<14%). These results demonstrate that BRBE significantly enhances the NER yields of a bulky and a non-bulky DNA lesion, and that this effect is correlated with an enhancement of expression of the critically important NER factor XPA and the helicase XPB, but not the helicase XPD.

Metabolic profiling workflow for cell extracts by targeted hydrophilic interaction liquid chromatography-tandem mass spectrometry

In this study, a targeted approach with wide metabolite coverage was developed for cellular metabolomic analysis using a UHPLC-QTrap-MS system operated in the scheduled multiple reaction monitoring (sMRM) mode. MRM ion pairs were acquired from HeLa cell samples through untargeted analysis using UHPLC-QTOF-MS with SWATH acquisition complemented by missing metabolites from pathway databases. Four different cell extraction protocols were studied and compared based on an experiment series involving the calculation of individual metabolite recoveries (pre/post extraction spiking U-13C isotope-labeled standards), with a Methanol/Water extraction mixture (1:1; v/v) showing the best results. Two HILIC-MS methods employing a Waters Premier BEH Amide column were developed, utilizing two different chromatographic conditions (20 mM ammonium formate as buffer additive adjusted to a pH = 3.5 with formic acid in ESI+ mode and 20 mM ammonium acetate adjusted to a pH = 7.5 with acetic acid in ESI− mode. One hundred sixty-one (161) metabolites were successfully detected in ESI+ mode, whereas 92 were detected in negative ionization mode, totaling to a number of 253 compounds in three different biological matrices covered by the analytical system employed. Both established HILIC methods were calibrated and validated based on 105 authentic chemical standards and U-13C-labeled Pichia pastoris (Komagataella phaffii) yeast extract as internal standards for cellular matrix (HeLa cells). Within-day and between-day precision was determined on three different QC concentration levels and was below 15% for the entirety of the analytes. Inter- and intra-day accuracies showed values in the range between 85 and 115% (assessed as % recovery) in the entire range. Matrix effects, extraction recoveries and process efficiencies were evaluated following the Matuszewski protocol with U-13C-labeled Pichia pastoris metabolite extract as internal standards. Eventually, the method was utilized to quantify metabolites in HeLa cell extracts.

Gp35/50 mucin molecules of Trypanosoma cruzi metacyclic forms that mediate host cell invasion interact with annexin A2.

Host cell invasion is a critical step for infection by Trypanosoma cruzi, the agent of Chagas disease. In natural infection, T. cruzi metacyclic trypomastigote (MT) forms establish the first interaction with host cells. The gp35/50 mucin molecules expressed in MT have been implicated in cell invasion process, but the mechanisms involved are not well understood. We performed a series of experiments to elucidate the mode of gp35/50-mediated MT internalization. Comparing two parasite strains from genetically divergent groups, G strain (TcI) and CL strain (TcVI), expressing variant forms of mucins, we demonstrated that G strain mucins participate in MT invasion. Only G strain-derived mucins bound to HeLa cells in a receptor-dependent manner and significantly inhibited G strain MT invasion. CL strain MT internalization was not affected by mucins from either strain. HeLa cell invasion by G strain MT was associated with actin recruitment and did not rely on lysosome mobilization. To examine the involvement of annexin A2, which plays a role in actin dynamic, annexin A2-depleted HeLa cells were generated. Annexin A2-deficient cell lines were significantly more resistant than wild type controls to G strain MT invasion. In a co-immunoprecipitation assay, to check whether annexin A2 might be the receptor for mucins, protein A/G magnetic beads crosslinked with monoclonal antibody to G strain mucins were incubated with detergent extracts of MT and HeLa cells. Binding of gp35/50 mucins to annexin A2 was detected. Both G strain MT and purified mucins induced focal adhesion kinase activation in HeLa cells. By confocal immunofluorescence microscopy, colocalization of invading G strain MT with clathrin was visualized. Inhibition of clathrin-coated vesicle formation reduced parasite internalization. Taken together, our data indicate that gp35/50-mediated MT invasion is accomplished through interaction with host cell annexin A2 and clathrin-dependent endocytosis.

A multianalyte assay for the detection of dermatomyositis-related autoantibodies based on immunoprecipitation combined with immunoblotting.

To develop a multianalyte assay for the detection of dermatomyositis (DM)-related autoantibodies using immunoprecipitation (IP) combined with immunoblotting (IB). Sera from 116 DM patients were subjected to RNA and protein immunoprecipitation assays as well as commercial enzyme-linked immunosorbent assays (ELISAs) for anti-aminoacyl transfer RNA synthetase, anti-melanoma differentiation antigen 5 (MDA5), anti-Mi-2, anti-transcriptional intermediary factor-1γ (TIF-1γ), and anti-U1 ribonucleoprotein antibodies. The IP/IB assay was developed by immunoprecipitation of autoantigens from HeLa cell extracts using patient sera, followed by immunoblotting with an antibody against Mi-2, TIF-1γ, OJ, nuclear matrix protein (NXP)-2, MDA5, PM/Scl, small ubiquitin-like modifier activating enzyme (SAE), or Ku. A multianalyte assay was designed by mixing primary antibodies in the IP/IB assay. IP assays identified any DM-related autoantibodies in 100 patients (86%), of which 82% were covered by commercial ELISAs, with a false-positive result in two sera and a false-negative result in one serum. The results obtained from the multianalyte IP/IB assay and 'gold-standard' IP assays were concordant in terms of the presence or absence of anti-MDA5, anti-TIF-1γ, anti-OJ, anti-NXP-2, anti-PM/Scl, anti-SAE, anti-Mi-2, and anti-Ku antibodies. This multianalyte IP/IB assay combined with commercial ELISAs is an alternative to 'gold-standard' IP assays for the detection of DM-related autoantibodies.

Role of ubiquitin-protein ligase UBR5 in the disassembly of mitotic checkpoint complexes

The mitotic (or spindle assembly) checkpoint system ensures accurate chromosome segregation in mitosis by preventing the onset of anaphase until correct bipolar attachment of sister chromosomes to the mitotic spindle is attained. It acts by promoting the assembly of a mitotic checkpoint complex (MCC), composed of mitotic checkpoint proteins BubR1, Bub3, Mad2, and Cdc20. MCC binds to and inhibits the action of ubiquitin ligase APC/C (anaphase-promoting complex/cyclosome), which targets for degradation regulators of anaphase initiation. When the checkpoint system is satisfied, MCCs are disassembled, allowing the recovery of APC/C activity and initiation of anaphase. Many of the pathways of the disassembly of the different MCCs have been elucidated, but the mode of their regulation remained unknown. We find that UBR5 (ubiquitin-protein ligase N-recognin 5) is associated with the APC/C*MCC complex immunopurified from extracts of nocodazole-arrested HeLa cells. UBR5 binds to mitotic checkpoint proteins BubR1, Bub3, and Cdc20 and promotes their polyubiquitylation invitro. The dissociation of a Bub3*BubR1 subcomplex of MCC is stimulated by UBR5-dependent ubiquitylation, as suggested by observations that this process in mitotic extracts requires UBR5 and α-β bond hydrolysis of adenosine triphosphate. Furthermore, a system reconstituted from purified recombinant components carries out UBR5- and ubiquitylation-dependent dissociation of Bub3*BubR1. Immunodepletion of UBR5 from mitotic extracts slows down the release of MCC components from APC/C and prolongs the lag period in the recovery of APC/C activity in the exit from mitotic checkpoint arrest. We suggest that UBR5 may be involved in the regulation of the inactivation of the mitotic checkpoint.

CRB-SWATH: A Method for Enhancing Untargeted Precursor Ion Extraction and Automatically Constructing Their Tandem Mass Spectra from SWATH Datasets by Chromatographic Retention Behaviors.

Sequential window acquisition of all theoretical spectra (SWATH) as a typical data-independent acquisition (DIA) strategy is favorable for untargeted metabolomics. It could theoretically acquire product ions of all precursor ions, including precursor ions showing chromatographic peaks of rather poor qualities. However, existing data processing methods present limited capabilities in capturing poor-quality peaks of precursor ions. Thus, although their product ions could be acquired, their precursor ions are absent. Here, we present a new strategy, chromatographic retention behavior-SWATH (CRB-SWATH), that could unbiasedly capture poor-quality peaks and provide high resolutions of multiplexed mass spectroscopy (MS/MS) spectra in SWATH datasets. CRB-SWATH monitors CRBs of SWATH-MS signals under a series of altered elution gradients. As signals of compounds differ from noise by showing CRBs, both the precursor and fragment ions are captured, while ignoring their peak qualities. Moreover, CRB-SWATH offers good chances to resolve highly multiplexed MS/MS spectra in SWATH datasets because precursor ions coeluted in a single elution gradient often present different CRBs. In the untargeted metabolic analysis of Hela cell extracts, CRB-SWATH showed the advantage in exclusively capturing 2645 ions of poor-quality peaks (i.e., tiny peaks, discontinuous ion traces, tailing peaks, zigzag peaks, etc.), accounting for 34.4% of all the untargeted precursor ions extracted. Therein, it is noteworthy that among 2116 negative ions detected in hydrophilic interaction liquid chromatography (HILIC) mode, 1284 poor-quality ion peaks (>60%) were exclusively captured by CRB-SWATH. As CRB-SWATH automatically captures a large sum of true ion peaks of poor qualities, extracts MS/MS spectra of high purities, and provides chromatographic retention behaviors of untargeted metabolites for identification and classification, it could be a useful metabolomics tool for understanding biological phenomena better.

Cytotoxic activity of cholesterol oxidase produced by Streptomyces sp. AKHSS against cancerous cell lines: mechanism of action in HeLa cells.

Re-occurrence of cancer is the major drawback for the currently available anticancer therapies. Therefore, study of an efficient enzyme, cholesterol oxidase produced by various kinds of microbes especially obtained from unexplored marine actinobacterial species against human cancer cell lines and understanding its mechanism of action helps to identify an irreversible and potent anticancer agent. The cytotoxic potential of cholesterol oxidase produced by a marine Streptomyces sp. AKHSS against four different human cancer cell lines was demonstrated through MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] assay. Fluorescent confocal microscopy and flow cytometry based experiments were performed to understand the efficiency of the enzymatic action on HeLa cells. Further, the apoptotic related proteins were detected through western blotting. Interestingly, the enzyme exhibited potent cytotoxicity at very low concentrations (0.093-0.327µM) against all the cells tested. Fluorescent confocal microscopy revealed the morphological variations induced by the enzyme on cancer cell lines such as the formation of lipid droplets and condensation of nuclei. The enzyme treated cell-free extracts of HeLa cells analyzed through gas chromatography mass spectrometry showed the depletion of membrane cholesterol and the presence of substituted enzyme oxidized product, cholest-4-ene-3-one. The enzyme had induced significant inhibitory effects on the cell viability such as cell cycle arrest (G1 phase), apoptosis and rise of reactive oxygen species as evident through flow cytometry. Besides, hyperpolarization of mitochondrial membrane, reduced rates of phosphorylation of pAkt and the expression of apoptotic death markers like Fas, Fas L, caspases (8 and 3) and PARP-1 were recorded in the enzyme treated HeLa cells. Thus, cholesterol oxidase purified from a marine Streptomyces sp. AKHSS exhibits potent cytotoxicity at very low concentrations against human cancer cell lines. All the ex vivo experiments portrayed the substantial inhibitory effect of the enzyme on HeLa cells suggesting that cholesterol oxidase of Streptomyces sp. AKHSS could be a prominent cancer chemotherapeutic agent.

Isomer Selective Comprehensive Lipidomics Analysis of Phosphoinositides in Biological Samples by Liquid Chromatography with Data Independent Acquisition Tandem Mass Spectrometry.

Phosphoinositides (PIPx) play central roles in membrane dynamics and signal transduction of key functions like cellular growth, proliferation, differentiation, migration, and adhesion. They are highly regulated through a network of distinct phosphatidylinositol phosphates consisting of seven groups and three regioisomers in two groups (PIP and PIP2), which arise from phosphorylation at 3', 4', and 5' positions of the inositol ring. Numerous studies have revealed the importance of both fatty acyl chains, degree of phosphorylation, and phosphorylation positions under physiological and pathological states. However, a comprehensive analytical method that allows differentiation of all regioisomeric forms with different acyl side chains and degrees of phosphorylation is still lacking. Here, we present an integrated comprehensive workflow of PIPx analysis utilizing a chiral polysaccharide stationary phase coupled with electrospray ionization high-resolution mass spectrometry with a data independent acquisition technique using the SWATH technology. Correspondingly, a targeted data mining strategy in the untargeted comprehensively acquired MS and MS/MS data was developed. This powerful highly selective method gives a full picture of PIPx profiles in biological samples. Herein, we present for the first time the full PIPx profiles of NIST SRM1950 plasma, Pichia pastoris lipid extract, and HeLa cell extract, including profile changes upon treatment with potential PI3K inhibitor wortmannin. We also illustrate using this inhibitor that measurements of the PIPx profile averaged over the distinct regioisomers by analytical procedures, which cannot differentiate between the individual PIPx isomers, can easily lead to biased conclusions.

Strategy for high-throughput identification of protein complexes by array-based multi-dimensional liquid chromatography-mass spectrometry

Comprehensive elucidation of the composition of multiprotein complexes in model organisms is essential to understand conserved biological systems, but large-scale mapping physical association networks is still challenging due to limited throughput of present methods. In this work, a strategy coupling array-based online two-dimensional liquid chromatography (array-based 2D-LC) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) was demonstrated for high throughput and in-depth identification of protein complexes from cultured human HeLa cell extracts. Mixed-bed ion-exchange column was employed as the first dimensional (1stD) separating mode and an array consisting of eight reversed phase columns was developed as the second dimensional (2ndD) mode. Taking advantage of array parallel strategy, this online system showed an 8-fold increase in throughput. After array-based online 2D-LC separation, altogether 256 × 2ndD fractions were collected for further LC-MS/MS analysis. Public databases of protein-protein interaction (PPI) and co-elution curves identified by LC-MS were applied to reconstruct the protein complexes. A rigorous inspection was operated by cataloging the protein complexes into chromatographic fractions to minimize the number of false positives. As result, a total number of 4,436 proteins were identified and 26,092 elution curves were graphed. A network consisting of 47,745 PPIs was established among 2,201 proteins and presented 1,530 putative protein complexes with high confidence. Most of the identified PPIs were linked to diverse biological processes and may reveal further disease mechanism and therapeutic strategy.

Base and Nucleotide Excision Repair Pathways in DNA Plasmids Harboring Oxidatively Generated Guanine Lesions.

The base and nucleotide excision repair pathways (BER and NER, respectively) are two major mechanisms that remove DNA lesions formed by the reactions of genotoxic intermediates with cellular DNA. We have demonstrated earlier that the oxidatively generated guanine lesions spiroiminodihydantoin (Sp) and 5-guanidinohydantoin (Gh) are excised from double-stranded DNA by competing BER and NER in whole-cell extracts [Shafirovich, V., et al. (2016) J. Biol. Chem. 321, 5309-5319]. In this work we compared the NER and BER yields with single Gh or Sp lesions embedded at the same sites in covalently closed circular pUC19NN plasmid DNA (cccDNA) and in the same but linearized form (linDNA) of this plasmid. The kinetics of the Sp and Gh BER and NER incisions were monitored in HeLa cell extracts. The yield of NER products is ∼5 times greater in covalently closed circular DNA than in the linearized form, while the BER yield is smaller by ∼20-30% depending on the guanine lesion. Control BER experiments with 8-oxo-7,8-dihydroguanine (8-oxoG) show that the BER yield is increased by a factor of only 1.4 ± 0.2 in cccDNA relative to linDNA. These surprising differences in BER and NER activities are discussed in terms of the lack of termini in covalently closed circular DNA and the DNA lesion search dynamics of the NER DNA damage sensor XPC-RAD23B and the BER enzyme OGG1 that recognizes and excises 8-oxoG.