A single step and rapid protein extraction protocol developed for cell lines and tissues: Compatible for gel based and gel free proteomic approaches.

Proteins are crucial research molecules in modern biology. Almost every biological research area needs protein-based assays to answer the research questions. The study of the total protein content of a biological sample known as Proteomics, is one of the highly rated qualitative and quantitative approach to address numerous biological problems including clinical research. The key step to successfully generate high quality proteomics data is the efficient extraction of proteins from biological samples. Although different methods are in use for protein extraction from a wide variety of samples, however, because of their prolonged protocol and multiple steps involved, final protein yield is sacrificed. Here, we have shown the development of a simple single step method for extraction of proteins from mammalian cell lines as well as tissue samples in an effective and reproducible manner. This method is based on lysis of samples directly in a modified lysis buffer without CHAPS (7 M Urea, 2 M Thiourea, and 10 mM Tris-Cl; pH 8.5) that is compatible with gel based and gel free approaches. This developed protocol is reliable and should be useful for a wide range of proteomic studies involving various biological samples.

Irreversible cell cycle exit associated with senescence is mediated by constitutive MYC degradation.

Cells can irreversibly exit the cell cycle and become senescent to safeguard against uncontrolled proliferation. While the p53-p21 and p16-Rb pathways are thought to mediate senescence, they also mediate reversible cell cycle arrest (quiescence), raising the question of whether senescence is actually reversible or whether alternative mechanisms underly the irreversibility associated with senescence. Here, we show that senescence is irreversible and that commitment to and maintenance of senescence are mediated by irreversible MYC degradation. Senescent cells start dividing when a non-degradable MYC mutant is expressed, and quiescent cells convert to senescence when MYC is knocked down. In early oral carcinogenesis, epithelial cells exhibit MYC loss and become senescent as a safeguard against malignant transformation. Later stages of oral premalignant lesions exhibit elevated MYC levels and cellular dysplasia. Thus, irreversible cell cycle exit associated with senescence is mediated by constitutive MYC degradation, but bypassing this degradation may allow tumor cells to escape during cancer initiation.

Feeding enhances fibronectin adherence of quiescent lymphocytes through non-canonical insulin signalling.

Nutritional availability during fasting and refeeding affects the temporal redistribution of lymphoid and myeloid immune cells among the circulating and tissue-resident pools. Conversely, nutritional imbalance and impaired glucose metabolism are associated with chronic inflammation, aberrant immunity and anomalous leukocyte trafficking. Despite being exposed to periodic alterations in blood insulin levels upon fasting and feeding, studies exploring the physiological effects of these hormonal changes on quiescent immune cell function and trafficking are scanty. Here, we report that oral glucose load in mice and healthy men enhances the adherence of circulating peripheral blood mononuclear cells (PBMCs) and lymphocytes to fibronectin. Adherence to fibronectin is also observed upon regular intake of breakfast following overnight fasting in healthy subjects. This glucose load-induced phenomenon is abrogated in streptozotocin-injected mice that lack insulin. Intra-vital microscopy in mice demonstrated that oral glucose feeding enhances the homing of PBMCs to injured blood vessels in vivo. Furthermore, employing flow cytometry, Western blotting and adhesion assays for PBMCs and Jurkat-T cells, we elucidate that insulin enhances fibronectin adherence of quiescent lymphocytes through non-canonical signalling involving insulin-like growth factor-1 receptor (IGF-1R) autophosphorylation, phospholipase C gamma-1 (PLCγ-1) Tyr783 phosphorylation and inside-out activation of ß-integrins respectively. Our findings uncover the physiological relevance of post-prandial insulin spikes in regulating the adherence and trafficking of circulating quiescent T-cells through fibronectin-integrin interaction.

Asymmetrically Coordinated Heterodimetallic Ir-Ru System: Synthesis, Computational, and Anticancer Aspects.

Herein, we present an unprecedented formation of a heterodinuclear complex [{(ppy)2IrIII}(µ-phpy){RuII(tpy)}](ClO4)2 {[1](ClO4)2} using terpyridyl/phenylpyridine as ancillary ligands and asymmetric phpy as a bridging ligand. The asymmetric binding mode (N∧N-∩-N∧N∧C-) of the phpy ligand in {[1](ClO4)2} is confirmed by 1H, 13C, 1H-1H correlated spectroscopy (COSY), high-resolution mass spectrum (HRMS), single-crystal X-ray crystallography techniques, and solution conductivity measurements. Theoretical investigation suggests that the highest occupied molecular orbital (HOMO) and the least unoccupied molecular orbital (LUMO) of [1]2+ are located on iridium/ppy and phpy, respectively. The complex displays a broad low energy charge transfer (CT) band within 450-575 nm. The time-dependent density functional theory (TDDFT) analysis suggests this as a mixture of metal-to-ligand charge transfer (MLCT) and ligand-to-ligand charge transfer (LLCT), where both ruthenium, iridium, and ligands are involved. Complex {[1](ClO4)2} exhibits RuIIIrIII/RuIIIIrIII- and RuIIIIrIII/RuIIIIrIV-based oxidative couples at 0.83 and 1.39 V, respectively. The complex shows anticancer activity and selectivity toward human breast cancer cells (IC50; MCF-7: 9.3 ± 1.2 µM, and MDA-MB-231: 8.6 ± 1.2 µM) over normal breast cells (MCF 10A: IC50 ≈ 21 ± 1.3 µM). The Western blot analysis and fluorescence microscopy images suggest that combined apoptosis and autophagy are responsible for cancer cell death.

Revealing ß-TrCP activity dynamics in live cells with a genetically encoded biosensor.

The F-box protein beta-transducin repeat containing protein (ß-TrCP) acts as a substrate adapter for the SCF E3 ubiquitin ligase complex, plays a crucial role in cell physiology, and is often deregulated in many types of cancers. Here, we develop a fluorescent biosensor to quantitatively measure ß-TrCP activity in live, single cells in real-time. We find ß-TrCP remains constitutively active throughout the cell cycle and functions to maintain discreet steady-state levels of its substrates. We find no correlation between expression levels of ß-TrCP and ß-TrCP activity, indicating post-transcriptional regulation. A high throughput screen of small-molecules using our reporter identifies receptor-tyrosine kinase signaling as a key axis for regulating ß-TrCP activity by inhibiting binding between ß-TrCP and the core SCF complex. Our study introduces a method to monitor ß-TrCP activity in live cells and identifies a key signaling network that regulates ß-TrCP activity throughout the cell cycle.

Ezrin gone rogue in cancer progression and metastasis: An enticing therapeutic target.

Cancer metastasis is the primary cause of morbidity and mortality in cancer as it remains the most complicated, devastating, and enigmatic aspect of cancer. Several decades of extensive research have identified several key players closely associated with metastasis. Among these players, cytoskeletal linker Ezrin (the founding member of the ERM (Ezrin-Radixin-Moesin) family) was identified as a critical promoter of metastasis in pediatric cancers in the early 21st century. Ezrin was discovered 40 years ago as a aminor component of intestinal epithelial microvillus core protein, which is enriched in actin-containing cell surface structures. It controls gastric acid secretion and plays diverse physiological roles including maintaining cell polarity, regulating cell adhesion, cell motility and morphogenesis. Extensive research for more than two decades evinces that Ezrin is frequently dysregulated in several human cancers. Overexpression, altered subcellular localization and/or aberrant activation of Ezrin are closely associated with higher metastatic incidence and patient mortality, thereby justifying Ezrin as a valuable prognostic biomarker in cancer. Ezrin plays multifaceted role in multiple aspects of cancer, with its significant contribution in the complex metastatic cascade, through reorganizing the cytoskeleton and deregulating various cellular signaling pathways. Current preclinical studies using genetic and/or pharmacological approaches reveal that inactivation of Ezrin results in significant inhibition of Ezrin-mediated tumor growth and metastasis as well as increase in the sensitivity of cancer cells to various chemotherapeutic drugs. In this review, we discuss the recent advances illuminating the molecular mechanisms responsible for Ezrin dysregulation in cancer and its pleiotropic role in cancer progression and metastasis. We also highlight its potential as a prognostic biomarker and therapeutic target in various cancers. More importantly, we put forward some potential questions, which we strongly believe, will stimulate both basic and translational research to better understand Ezrin-mediated malignancy, ultimately leading to the development of Ezrin-targeted cancer therapy for the betterment of human life.

Nuclear pore protein NUP210 depletion suppresses metastasis through heterochromatin-mediated disruption of tumor cell mechanical response.

Mechanical signals from the extracellular microenvironment have been implicated in tumor and metastatic progression. Here, we identify nucleoporin NUP210 as a metastasis susceptibility gene for human estrogen receptor positive (ER+) breast cancer and a cellular mechanosensor. Nup210 depletion suppresses lung metastasis in mouse models of breast cancer. Mechanistically, NUP210 interacts with LINC complex protein SUN2 which connects the nucleus to the cytoskeleton. In addition, the NUP210/SUN2 complex interacts with chromatin via the short isoform of BRD4 and histone H3.1/H3.2 at the nuclear periphery. In Nup210 knockout cells, mechanosensitive genes accumulate H3K27me3 heterochromatin modification, mediated by the polycomb repressive complex 2 and differentially reposition within the nucleus. Transcriptional repression in Nup210 knockout cells results in defective mechanotransduction and focal adhesion necessary for their metastatic capacity. Our study provides an important role of nuclear pore protein in cellular mechanosensation and metastasis.

Cell cycle inertia underlies a bifurcation in cell fates after DNA damage.

The G1-S checkpoint is thought to prevent cells with damaged DNA from entering S phase and replicating their DNA and efficiently arrests cells at the G1-S transition. Here, using time-lapse imaging and single-cell tracking, we instead find that DNA damage leads to highly variable and divergent fate outcomes. Contrary to the textbook model that cells arrest at the G1-S transition, cells triggering the DNA damage checkpoint in G1 phase route back to quiescence, and this cellular rerouting can be initiated at any point in G1 phase. Furthermore, we find that most of the cells receiving damage in G1 phase actually fail to arrest and proceed through the G1-S transition due to persistent cyclin-dependent kinase (CDK) activity in the interval between DNA damage and induction of the CDK inhibitor p21. These observations necessitate a revised model of DNA damage response in G1 phase and indicate that cells have a G1 checkpoint.

Proteomics and functional study reveal marginal zone B and B1 cell specific protein as a candidate marker of multiple myeloma.

Multiple myeloma (MM) is a plasma cell­associated cancer and accounts for 13% of all hematological malignancies, worldwide. MM still remains an incurable plasma cell malignancy with a poor prognosis due to a lack of suitable markers. Therefore, discovering novel markers and targets for diagnosis and therapeutics of MM is essential. The present study aims to identify markers associated with MM malignancy using patient­derived MM mononuclear cells (MNCs). Label­free quantitative proteomics analysis revealed a total of 192 differentially regulated proteins, in which 79 proteins were upregulated and 113 proteins were found to be downregulated in MM MNCs as compared to non­hematological malignant samples. The identified differentially expressed candidate proteins were analyzed using various bioinformatics tools, including Ingenuity Pathway Analysis (IPA), Protein Analysis THrough Evolutionary Relationships (PANTHER), Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) and Database for Annotation, Visualization and Integrated Discovery (DAVID) to determine their biological context. Among the 192 candidate proteins, marginal zone B and B1 cell specific protein (MZB1) was investigated in detail using the RPMI-8226 cell line model of MM. The functional studies revealed that higher expression of MZB1 is associated with promoting the progression of MM pathogenesis and could be established as a potential target for MM in the future.

XPO1 is a critical player for bortezomib resistance in multiple myeloma: A quantitative proteomic approach.

Among the blood cancers, 13% mortality is caused by Multiple myeloma (MM) type of hematological malignancy. In spite of therapeutic advances in chemotherapy treatment, still MM remains an incurable disease is mainly due to emergence of chemoresistance. At present time, FDA approved bortezomib is the first line drug for MM treatment. However, like other chemotherapy, MM patients are acquiring resistance against bortezomib. The present study aims to identify and validate bortezomib resistant protein targets in MM using iTRAQ and label free quantitative proteomic approaches. 112 differentially expressed proteins were commonly found in both approaches with similar differential expression pattern. Exportin-1 (XPO1) protein was selected for further validation as its significant high expression was observed in both iTRAQ and label free analysis. Bioinformatic analysis of these common differentially expressed proteins showed a clear cluster of proteins such as SMC1A, RCC2, CSE1, NUP88, NUP50, TPR, HSPA14, DYNLL1, RAD21 and RANBP2 being associated with XPO1. Functional studies like cell count assay, flow cytometry assay and soft agar assay proved that XPO1 knock down in RPMI 8226R cell line results in re-sensitization to bortezomib drug. The mass spectrometry data are available via ProteomeXchange with identifier PXD013859. BIOLOGICAL SIGNIFICANCE: Multiple myeloma (MM) is a type of hematological malignancy which constitutes about 13% of all blood cell related malignancies. Chemoresistance is one of the major obstacles for the successful treatment for MM. Bortezomib is a first proteasome inhibitor drug, widely used in MM treatment. The present study aims to identify and validate bortezomib resistant protein targets in MM. Here, we identified 112 candidate proteins to be associated with bortezomib resistance using global quantitative proteomic analysis. Among these candidate proteins, we show that XPO1 plays crucial role in emerging bortezomib resistance using functional studies like cell count assay, flow cytometry assay and soft agar assay. XPO1 could be a potential therapeutic target for MM and development of inhibitors of XPO1 might help to cure MM.

F-box protein FBXO16 functions as a tumor suppressor by attenuating nuclear ß-catenin function.

Aberrant activation of ß-catenin has been implicated in a variety of human diseases, including cancer. In spite of significant progress, the regulation of active Wnt/ß-catenin-signaling pathways is still poorly understood. In this study, we show that F-box protein 16 (FBXO16) is a putative tumor suppressor. It is a component of the SCF (SKP1-Cullin1-F-box protein) complex, which targets the nuclear ß-catenin protein to facilitate proteasomal degradation through the 26S proteasome. FBXO16 interacts physically with the C-terminal domain of ß-catenin and promotes its lysine 48-linked polyubiquitination. In addition, it inhibits epithelial-to-mesenchymal transition (EMT) by attenuating the level of ß-catenin. Therefore, depletion of FBXO16 leads to increased levels of ß-catenin, which then promotes cell invasion, tumor growth, and EMT of cancer cells. Furthermore, FBXO16 and ß-catenin share an inverse correlation of cellular expression in clinical breast cancer patient samples. In summary, we propose that FBXO16 functions as a putative tumor suppressor by forming an SCFFBXO16 complex that targets nuclear ß-catenin in a unique manner for ubiquitination and subsequent proteasomal degradation to prevent malignancy. This work suggests a novel therapeutic strategy against human cancers related to aberrant ß-catenin activation. © 2019 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Protein Phosphatase 1 Regulatory Subunit SDS22 Inhibits Breast Cancer Cell Tumorigenesis by Functioning as a Negative Regulator of the AKT Signaling Pathway.

Protein phosphatases play a crucial role in cell cycle progression, cell survival, cellular signaling, and genomic integrity. The protein phosphatase 1 (PP1) regulatory subunit SDS22 plays a significant role in cell cycle progression. A recent study showed that SDS22 plays a vital role in epithelial integrity and tumor suppression in Drosophila. However, its tumor suppressive activity remains obscure in the mammalian system. Here, for the first time, we show that SDS22 inhibits the growth of breast cancer cells through induction of apoptosis. SDS22 negatively regulates the AKT kinase signaling pathway through PP1. SDS22 associates predominantly with AKT and dephosphorylates the phospho Thr308 and phospho Ser473 through PP1 and hence abrogates the cell migration, invasion, and tumor growth. Thus, our study deciphers the long-standing question of how PP1 negatively regulates the AKT signaling pathway. Further, we observed a significant converse correlation in the expression levels of SDS22 and phospho form of AKT with reduced levels of SDS22 in the higher grades of cancer. Overall, our results suggest that SDS22 could be a putative tumor suppressor and replenishment of SDS22 would be an important strategy to restrict the tumor progression.

FBXO32 activates NF-κB through IκBα degradation in inflammatory and genotoxic stress.

In response to diverse stresses, the canonical NF-κB pathway gets activated primarily to protect the cells and maintain their genomic integrity. It activates the cell cycle checkpoints allowing the cells with limited damage to restore a normal life cycle. One of the key events in activation of the canonical NF-κB pathway is the selective proteasomal degradation of IκBα. It has been previously shown that F-box protein ßTRCP1 has limited role in directing the proteasomal degradation of IκBα during stress conditions. Here, we report another member of F-box family proteins, FBXO32, as a potential activator of NF-κB signaling during genotoxic stress and inflammatory response. Following genotoxic or inflammatory stress, FBXO32 is stabilized, which leads to polyubiquitination and proteasome mediated degradation of IκBα. We also found that FBXO32 is required for physiological regulation of IκBα levels in unstressed cells. Thus, we decipher the new role of FBXO32 in regulation of NF-κB signaling pathway.

Cdc20 directs proteasome-mediated degradation of the tumor suppressor SMAR1 in higher grades of cancer through the anaphase promoting complex.

The Tumor suppressor SMAR1 (scaffold matrix attachment region binding protein 1) has a crucial role in maintaining genomic stability, cell cycle progression and apoptosis.Our previous finding showed that it is highly suppressed in higher grade of cancer. However, the underlying mechanism of this suppression was not well understood. In this study, we show that SMAR1 expression levels are controlled at the proteasomal level by five RING finger E3 ubiquitin ligases including, Cdc20, a substrate receptor of ubiquitin ligase APC/C complex. We found that Cdc20 binds and promotes proteasomal degradation of SMAR1 in a D-box motif dependent manner. Further, our results demonstrated that Cdc20 promotes proteasomal degradation of SMAR1 through K48-linked specific polyubiquitylation, and that short hairpin RNA mediated inactivation of Cdc20 leads to significant stabilization of SMAR1. These findings suggest that Cdc20 is responsible for maintaining the cellular levels of SMAR1. However, since Cdc20 fails to target SMAR1 upon exposure to genotoxic stresses, SMAR1 helps to maintain genomic stability under these conditions through its DNA damage repair activity. Interestingly, Cdc20-mediated degradation of SMAR1 promotes cell migration and invasion.The reciprocal relationship of the duo is evident in breast cancer cell lines as well as in patient samples, suggesting that Cdc20 functions as an important negative regulator of SMAR1 in higher grades of cancer. Our study reveals for the first time, the molecular mechanism associated with lower levels of expression of the important tumor suppressor SMAR1 in higher grades of breast cancer.

A MicroRNA/Ubiquitin Ligase Feedback Loop Regulates Slug-Mediated Invasion in Breast Cancer.

The transformation of a normal cell to cancer requires the derail of multiple pathways. Normal signaling in a cell is regulated at multiple stages by the presence of feedback loops, calibration of levels of proteins by their regulated turnover, and posttranscriptional regulation, to name a few. The tumor suppressor protein FBXO31 is a component of the SCF E3 ubiquitin ligase and is required to arrest cells at G1 following genotoxic stresses. Due to its growth-suppression activity, it is underexpressed in many cancers. However, the molecular mechanism underlying the translational regulation of FBXO31 remains unclear. Here we show that the oncogenic microRNAs miR-93 and miR-106a repress FBXO31, resulting in the upregulation of Slug, which is involved in epithelial-mesenchymal transition and cell invasion. FBXO31 targets and ubiquitylates Slug for proteasomal degradation. However, this mechanism is repressed in breast tumors where miR-93 and miR-106a are overexpressed. Our study further unravels an interesting mechanism whereby Slug drives the expression of miR-93 and miR-106a, thus establishing a positive feedback loop to maintain an invasive phenotype. Together, these results establish the presence of interplay between microRNAs and the ubiquitination machinery, which together regulate cancer cell invasion.

Global proteomic profiling identifies etoposide chemoresistance markers in non-small cell lung carcinoma.

Chemoresistance is one of the leading health concerns in cancer treatment. Understanding the mechanism of chemoresistance is the best way to improve the survival of the patient. Etoposide and its analogues are widely used as antitumor drugs in lung cancer but many etoposide resistant lung cancer cases has been identified in recent years. The present study aims to explore the cellular response of lung cancer cell lines to etoposide and finding the potential chemoresistant marker proteins. Multiple proteomic platforms like 2-DE, DIGE and iTRAQ have been used to study the global proteome profile of NCI-H460 and etoposide resistant NCI-H460R cell lines. Our study revealed that etoposide treatment leads to alteration of 83 proteins in NCI-H460R cell lines. The functional analysis highlighted the role of the differential expressed proteins in cellular signaling, apoptosis, and cytoskeleton reorganization. Our study has identified several new proteins like RHOC, DLG5, UGDH, TMOD3 in addition to known chemoresistance associated proteins. In silico prediction of the important selected candidates are further validated at protein and mRNA level. Further, functional studies of newly identified candidate genes RHOC and DLG5 revealed that chemotherapeutic resistance is associated with their elevated level and may serve as novel targets for therapeutic intervention. BIOLOGICAL SIGNIFICANCE: Etoposide and its analogues have been used for lung cancer treatment for a while and it was reported that many non small cell lung carcinoma patients are resistant to etoposide. Although etoposide show drug resistance, the exact mechanism was not well understood. The present study focused on the global proteome analysis of NCI-H460 and NCI-H460R cell lines using multiple proteomic platforms to understand the potential chemoresistant markers for etoposide. Our multi-proteomic analysis has showed differential expression of 83 proteins involved in oxidative phosphorylation, metabolic, protein folding, cytoskeleton associated protein along with apoptotic pathway has been identified. In addition, quite a few interesting proteins such as RHOC, DLG5, HSP90, citrate synthase, UDP-glucose-6-dehydrogenase, Tropomodulin-3 are involved in chemoresistance has been observed. Overall, this is the first comprehensive proteomic study on etoposide resistant cell line NCI-H460 to explore the mechanism of chemoresistance in lung cancer.

Mechanistic insights into the phosphatidylinositol binding properties of the pleckstrin homology domain of lamellipodin.

Lamellipodin (Lpd) protein plays an important role in the formation of lamellipodial protrusion which is crucial in actin dynamics, cell polarity and motility. Lpd promotes actin polymerization with the help of members of the Ena/VASP family of actin regulators and tethering them to actin filaments. It is well documented that Lpd protein interacts with the membrane containing phosphatidylinositols through its pleckstrin homology (PH) domain and regulates several cellular functions and cell migration. However, the molecular mechanism that underlies how the PH domain of Lpd specifically gets recruited to phosphatidylinositols remains unclear. To understand their interaction properties, we quantitatively determined the binding parameters of the Lpd-PH domain employing a number of biophysical studies including surface plasmon resonance (SPR), fluorescence resonance energy transfer (FRET)-based competitive binding assay and monolayer penetration measurements. Our studies showed that the Lpd-PH domain strongly interacts with PI(3,4)P2 containing liposome without any membrane penetration. Mutational studies demonstrate that the presence of cationic residues within the phosphatidylinositol (PIP) binding site of the Lpd-PH domain is essential in membrane binding. The translocation patterns of the Lpd-PH domain and mutants in platelet-derived growth factor (PDGF) stimulated A549 cells are in good agreement with our in vitro binding measurements. Overall, these studies demonstrate an insight into how the Lpd-PH domain regulates cellular signals in a PI(3,4)P2 dependent manner.

Insights into the inhibitory mechanism of triazole-based small molecules on phosphatidylinositol-4,5-bisphosphate binding pleckstrin homology domain.

BACKGROUND: Phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] is an important regulator of several cellular processes and a precursor for other second messengers which are involved in cell signaling pathways. Signaling proteins preferably interact with PI(4,5)P2 through its pleckstrin homology (PH) domain. Efforts are underway to design small molecule-based antagonist, which can specifically inhibit the PI(4,5)P2/PH-domain interaction to establish an alternate strategy for the development of drug(s) for phosphoinositide signaling pathways. METHODS: Surface plasmon resonance, molecular docking, circular dichroism, competitive Förster resonance energy transfer, isothermal titration calorimetric analyses and liposome pull down assay were used. RESULTS: In this study, we employed 1,2,3-triazol-4-yl methanol containing small molecule (CIPs) as antagonists for PI(4,5)P2/PH-domain interaction and determined their inhibitory effect by using competitive-surface plasmon resonance analysis (IC50 ranges from 53 to 159 nM for PI(4,5)P2/PLCδ1-PH domain binding assay). We also used phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P3], phosphatidylinositol 3,4-bisphosphate [PI(3,4)P2], PI(4,5)P2 specific PH-domains to determine binding selectivity of the compounds. Various physicochemical analyses showed that the compounds have weak affect on fluidity of the model membrane but, strongly interact with the phospholipase C δ1 (PLCδ1)-PH domains. The 1,2,3-triazol-4-yl methanol moiety and nitro group of the compounds are essential for their exothermic interaction with the PH-domains. Potent compound can efficiently displace PLCδ1-PH domain from plasma membrane to cytosol in A549 cells. CONCLUSIONS: Overall, our studies demonstrate that these compounds interact with the PIP-binding PH-domains and inhibit their membrane recruitment. GENERAL SIGNIFICANCE: These results suggest specific but differential binding of these compounds to the PLCδ1-PH domain and emphasize the role of their structural differences in binding parameters. These triazole-based compounds could be directly used/further developed as potential inhibitor for PH domain-dependent enzyme activity.

Correlation of hydrogen-bonding propensity and anticancer profile of tetrazole-tethered combretastatin analogues.

A series of 1,5-disubstituted tetrazole-tethered combretastatin analogues with extended hydrogen-bond donors at the ortho-positions of the aryl A and B rings were developed and evaluated for their antitubulin and antiproliferative activity. We wanted to test whether intramolecular hydrogen-bonding used as a conformational locking element in these analogues would improve their activity. The correlation of crystal structures with the antitubulin and antiproliferative profiles of the modified analogues suggested that hydrogen-bond-mediated conformational control of the A ring is deleterious to the bioactivity. In contrast, although there was no clear evidence that intramolecular hydrogen bonding to the B ring enhanced activity, we found that increased substitution on the B ring had a positive effect on antitubulin and antiproliferative activity. Among the various analogues synthesized, compounds 5d and 5e, having hydrogen-bonding donor groups at the ortho and meta-positions on the 4-methoxy phenyl B ring, are strong inhibitors of tubulin polymerization and antiproliferative agents having IC50 value in micromolar concentrations.

Mass spectrometry-based proteomics in molecular diagnostics: discovery of cancer biomarkers using tissue culture.

Accurate diagnosis and proper monitoring of cancer patients remain a key obstacle for successful cancer treatment and prevention. Therein comes the need for biomarker discovery, which is crucial to the current oncological and other clinical practices having the potential to impact the diagnosis and prognosis. In fact, most of the biomarkers have been discovered utilizing the proteomics-based approaches. Although high-throughput mass spectrometry-based proteomic approaches like SILAC, 2D-DIGE, and iTRAQ are filling up the pitfalls of the conventional techniques, still serum proteomics importunately poses hurdle in overcoming a wide range of protein concentrations, and also the availability of patient tissue samples is a limitation for the biomarker discovery. Thus, researchers have looked for alternatives, and profiling of candidate biomarkers through tissue culture of tumor cell lines comes up as a promising option. It is a rich source of tumor cell-derived proteins, thereby, representing a wide array of potential biomarkers. Interestingly, most of the clinical biomarkers in use today (CA 125, CA 15.3, CA 19.9, and PSA) were discovered through tissue culture-based system and tissue extracts. This paper tries to emphasize the tissue culture-based discovery of candidate biomarkers through various mass spectrometry-based proteomic approaches.