Multi-carbon dots and aptamer based signal amplification ratiometric fluorescence probe for protein tyrosine kinase 7 detection.

BACKGROUND: Protein tyrosine kinase 7 (PTK 7) is a membrane receptor, which can be found in various kinds of cancers. In view of this, detection of PTK 7 in the peripheral circulation would be an effective way for the early diagnosis of cancer. RESULTS: In this work, a multi-carbon dots and aptamer-based signal amplification ratiometric fluorescence probe was developed. The fluorescence of the aptamer-modified y-CDs and b-CDs were respectively chosen as the detection signal and interior label. The fluorescence of y-CDs was quenched by Fe3O4 and cDNA (complement to aptamer) compound without PTK 7, but recovered by the addition of PTK 7. Then, the free aptamer was cut by DNase I, which amplified the detection signal. The ratiometric fluorescence sensor for PTK 7 was established with the LOD of 0.016 ng mL-1. CONCLUSIONS: Summary, a multi-carbon dots and aptamer-based signal amplification ratiometric fluorescence probe was developed for the detection of protein tyrosine kinase 7. The developed probe was applied to PTK 7 detection in MCF-7 cells and human serum with satisfying results, thus indicating that this probe has huge potential in clinical practice.

ROS1-fusion protein induces PD-L1 expression via MEK-ERK activation in non-small cell lung cancer.

Introduction: Despite some of the oncogenic driver mutations that have been associated with increased expression of programmed death-ligand 1 (PD-L1), the correlation between PD-L1 expression and ROS1 fusion in NSCLC cells, especially for those with Crizotinib resistance has not been fully addressed. Materials and Methods: The expression of PD-L1 in 30 primary NSCLC tumors with/without ROS1-fusion protein was evaluated by immunohistochemical (IHC) analysis. To assess the correlation between ROS1 fusion and PD-L1 expression, we down-regulated ROS1 with RNA interference or specific inhibitor (Crizotinib) in ROS1-fusion positive NSCLC cell line HCC78; or up-regulate ROS1-fusion gene in an immortalized human bronchial epithelial cell line (HBE). Mouse xenograft models were also used to determine the effect of ROS1 expression on PD-L1 expression in vivo. Crizotinib-resistant cell line was generated for measuring the association between Crizotinib resistance and PD-L1 expression. Results: ROS1-rearrangement in primary NSCLC tumor was significantly associated with up-regulated PD-L1 expression. PD-L1 expression was significantly up-regulated in bronchial epithelial cells after forced expression of ROS1 fusion and was eliminated when HCC78 xenograft mouse models were treated with Crizotinib. We found PD-L1 expression was modulated by MEK-ERK pathway signaling in both parental and Crizotinib-resistant NSCLC cells with ROS1 fusion. Conclusions: The correlation between ROS1-fusion and PD-L1 overexpression suggested that PD-L1/PD-1 blockade could be the second-line treatment option for the Crizotinib-resistant NSCLC with ROS1 rearrangement.

Immunopharmacology and Quantitative Analysis of Tyrosine Kinase Signaling.

In this article we describe the use of pharmacological and genetic tools coupled with immunoblotting (Western blotting) and targeted mass spectrometry to quantify immune signaling and cell activation mediated by tyrosine kinases. Transfer of the ATP γ phosphate to a protein tyrosine residue activates signaling cascades regulating the differentiation, survival, and effector functions of all cells, with unique roles in immune antigen receptor, polarization, and other signaling pathways. Defining the substrates and scaffolding interactions of tyrosine kinases is critical for revealing and therapeutically manipulating mechanisms of immune regulation. Quantitative analysis of the amplitude and kinetics of these effects is becoming ever more accessible experimentally and increasingly important for predicting complex downstream effects of therapeutics and for building computational models. Secondarily, quantitative analysis is increasingly expected by reviewers and journal editors, and statistical analysis of biological replicates can bolster claims of experimental rigor and reproducibility. Here we outline methods for perturbing tyrosine kinase activity in cells and quantifying protein phosphorylation in lysates and immunoprecipitates. The immunoblotting techniques are a guide to probing the dynamics of protein abundance, protein-protein interactions, and changes in post-translational modification. Immunoprecipitated protein complexes can also be subjected to targeted mass spectrometry to probe novel sites of modification and multiply modified or understudied proteins that cannot be resolved by immunoblotting. Together, these protocols form a framework for identifying the unique contributions of tyrosine kinases to cell activation and elucidating the mechanisms governing immune cell regulation in health and disease. © 2020 The Authors. Basic Protocol 1: Quantifying protein phosphorylation via immunoblotting and near-infrared imaging Alternate Protocol: Visualizing immunoblots using chemiluminescence Basic Protocol 2: Enriching target proteins and isolation of protein complexes by immunoprecipitation Support Protocol: Covalent conjugation of antibodies to functionalized beads Basic Protocol 3: Quantifying proteins and post-translational modifications by targeted mass spectrometry.

Expression, purification and crystallization of CLK1 kinase - A potential target for antiviral therapy.

Cdc-like kinase 1 (CLK1) is a dual-specificity kinase capable of autophosphorylation on tyrosine residues and Ser/Thr phosphorylation of its substrates. CLK1 belongs to the CLK kinase family that regulates alternative splicing through phosphorylation of serine-arginine rich (SR) proteins. Recent studies have demonstrated that CLK1 has an important role in the replication of influenza A and chikungunya viruses. Furthermore, CLK1 was found to be relevant for the replication of HIV-1 and the West Nile virus, making CLK1 an interesting cellular candidate for the development of a host-directed antiviral therapy that might be efficient for treatment of newly emerging viruses. We describe here our attempts and detailed procedures to obtain the recombinant kinase domain of CLK1 in suitable amounts for crystallization in complex with specific inhibitors. The key solution for the reproducibility of crystals resides in devising and refining expression and purification protocols leading to homogeneous protein. Co-expression of CLK1 with λ-phosphatase and careful purification has yielded crystals of CLK1 complexed with the KH-CB19 inhibitor that diffracted to 1.65 Å. These results paved the path to the screening of more structures of CLK1 complexed compounds, leading to further optimization of their inhibitory activity. Moreover, since kinases are desired targets in numerous pathologies, the approach we report here, the co-expression of kinases with λ-phosphatase, previously used in other kinases, can be adopted as a general protocol in numerous kinase targets for obtaining reproducible and homogenic non-phosphorylated (inactive) forms suitable for biochemical and structural studies thus facilitating the development of novel inhibitors.

Expression, purification, and characterization of the native intracellular domain of human epidermal growth factor receptors 1 and 2 in Escherichia coli.

Human epidermal growth factor receptors (EGFR) are an important target in drug discovery in terms of both protein-small-molecule interactions and protein-protein interactions. In this work, the isolation of a stable soluble protein of the tyrosine kinase domain of EGFR in Escherichia coli expression has been accomplished. This successful study presents the expression and purification conditions to obtain a stable soluble protein of the active tyrosine kinase domain of EGFR (EGFR-TK) and ErbB2 (ErbB2-TK) in a bacterial system, albeit in relatively low yields. The recombinant gene was inserted into a pColdI vector and recombinant protein was expressed at low temperature. Purification of EGFR-TK and ErbB2-TK took place under the same conditions by purified supernatant using a diethylaminoethyl sepharose column followed by anion exchange and size-exclusion chromatography columns. The final yields of purified EGFR-TK and ErbB2-TK were 8.4 and 9.5 mg per liter of culture, respectively. Determination of EGFR-TK and ErbB2-TK was performed via enzyme activity with commercial drugs. The IC50 values of erlotinib and afatinib against EGFR-TK were 13.09 nM and 2.36 nM respectively, while the IC50 values of lapatinib and afatinib against ErbB2-TK were 24.69 nM and 1.36 nM, respectively. These results confirmed that soluble proteins of the active intracellular domain of the HERs family were successfully expressed and purified in a bacterial system. The new protein expression and purification protocol will greatly facilitate the enzymatic inhibition and structural studies of this protein for drug discovery.

Synthesis and in vitro evaluation of diverse heterocyclic diphenolic compounds as inhibitors of DYRK1A.

Dual-specificity tyrosine phosphorylation-related kinase 1A (DYRK1A) is a dual-specificity protein kinase that catalyses phosphorylation and autophosphorylation. Higher DYRK1A expression correlates with cancer, in particular glioblastoma present within the brain. We report here the synthesis and biological evaluation of new heterocyclic diphenolic derivatives designed as novel DYRK1A inhibitors. The generation of these heterocycles such as benzimidazole, imidazole, naphthyridine, pyrazole-pyridines, bipyridine, and triazolopyrazines was made based on the structural modification of the lead DANDY and tested for their ability to inhibit DYRK1A. None of these derivatives showed significant DYRK1A inhibition but provide valuable knowledge around the importance of the 7-azaindole moiety. These data will be of use for developing further structure-activity relationship studies to improve the selective inhibition of DYRK1A.

Purification, characterization, and crystallization of membrane bound Escherichia coli tyrosine kinase.

Escherichia coli tyrosine kinase (Etk) is a membrane bound kinase in gram-negative bacteria that regulates the export of capsular polysaccharides (CPS). The molecular mechanism behind CPS regulation remains unclear, despite access to a crystal structure of the cytoplasmic kinase domain of Etk. In this study, an efficient protocol to produce full length Etk solubilized in n-dodecyl-ß-d-maltoside has been established with high yield. We have determined that detergent solubilized Etk retains kinase activity, but the protein is prone to aggregation, degradation, and has been unsuccessful in protein crystallization trials. In response, we designed and characterized truncations of Etk that do not aggregate and have led to successful crystallization experiments. In this article, we discuss our optimized expression and purification protocol for Etk, the design of Etk protein truncations, and the behavior of Etk during purification in a range of stabilizing detergents. These efforts have successfully produced protein suitable for crystallization. Our results will be a useful guide for future structural and functional studies of the bacterial tyrosine kinase family.

Drug Discovery of Host CLK1 Inhibitors for Influenza Treatment.

The rapid evolution of influenza virus makes antiviral drugs less effective, which is considered to be a major bottleneck in antiviral therapy. The key proteins in the host cells, which are related with the replication cycle of influenza virus, are regarded as potential drug targets due to their distinct advantage of lack of evolution and drug resistance. Cdc2-like kinase 1 (CLK1) in the host cells is responsible for alternative splicing of the M2 gene of influenza virus during influenza infection and replication. In this study, we carried out baculovirus-mediated expression and purification of CLK1 and established a reliable screening assay for CLK1 inhibitors. After a virtual screening of CLK1 inhibitors was performed, the activities of the selected compounds were evaluated. Finally, several compounds with strong inhibitory activity against CLK1 were discovered and their in vitro anti-influenza virus activities were validated using a cytopathic effect (CPE) reduction assay. The assay results showed that clypearin, corilagin, and pinosylvine were the most potential anti-influenza virus compounds as CLK1 inhibitors among the compounds tested. These findings will provide important information for new drug design and development in influenza treatment, and CLK1 may be a potent drug target for anti-influenza drug screening and discovery.

A novel protein tyrosine kinase Tec identified in lamprey, Lampetra japonica.

Protein tyrosine kinase Tec, a kind of non-receptor tyrosine kinase, is primarily found to be expressed in T cells, B cells, hematopoietic cells, and liver cells as a cytoplasmic protein. Tec has been proved to be a critical modulator of T cell receptor signaling pathway. In the present study, a homolog of Tec was identified in the lamprey, Lampetra japonica. The full-length Tec cDNA of L. japonica (Lja-Tec) contains a 1923 bp open reading frame that encodes a 641-amino acid protein. The multi-alignment of the deduced amino acid sequence of Lja-Tec with typical vertebrate Tecs showed that it possesses all conserved domains of the Tec family proteins, indicating that an ortholog of Tec exists in the extant jawless vertebrate. In the phylogenetic tree that was reconstructed with 24 homologs of jawless and jawed vertebrates, the Tecs from lampreys and hagfish were clustered as a single clade. The genetic distance between the outgroup and agnathan Tecs' group is closer than that between outgroup and gnathostome Tecs' group, indicating that its origin was far earlier than any of the jawed vertebrates. The mRNA levels of Lja-Tec in lymphocyte-like cells and gills were detected by real-time quantitative polymerase chain reaction. Results showed that it was significantly upregulated under stimulation with mixed pathogens. This result was further confirmed by western blot analysis. All these results indicated that Lja-Tec plays an important role in immune response. Our data will provide a reference for the further study of lamprey Tec and its immunological function in jawless vertebrates.

Characterization of the wzc gene from Pantoea sp. strain PPE7 and its influence on extracellular polysaccharide production and virulence on Pleurotus eryngii.

To characterize of the pathogenicity gene from the soft rot pathogen Pantoea sp. PPE7 in Pleurotus eryngii, we constructed over 10,000 kanamycin-resistant transposon mutants of Pantoea sp. strain PPE7 by transposon mutagenesis. One mutant, Pantoea sp. NPPE9535, did not cause a soft rot disease on Pleurotus eryngii was confirmed by the pathogenicity test. The transposon was inserted into the wzc gene and the disruption of the wzc gene resulted in the reduction of polysaccharide production and abolished the virulence of Pantoea sp. strain PPE7 in P. eryngii. Analysis of the hydropathic profile of this protein indicated that it is composed of two main domains: an N-terminal domain including two transmembrane α-helices and a C-terminal cytoplasmic domain consisting of a tyrosine-rich region. Comparative analysis indicated that the amino acid sequence of Wzc is similar to that of a number of proteins involved in the synthesis or export of polysaccharides in other bacterial species. Purified GST-Wzc was found to affect the phosphorylation of tyrosine residue in vivo. These results showed that the wzc gene might play an important role in the virulence of Pantoea sp. strain PPE7 in P. eryngii.

Co-expression of protein tyrosine kinases EGFR-2 and PDGFRß with protein tyrosine phosphatase 1B in Pichia pastoris.

The regulation of protein tyrosine phosphorylation is mediated by protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs) and is essential for cellular homeostasis. Coexpression of PTKs with PTPs in Pichia pastoris was used to facilitate the expression of active PTKs by neutralizing their apparent toxicity to cells. In this study, the gene encoding phosphatase PTP1B with or without a blue fluorescent protein or peroxisomal targeting signal 1 was cloned into the expression vector pAG32 to produce four vectors. These vectors were subsequently transformed into P. pastoris GS115. The tyrosine kinases EGFR-2 and PDGFRß were expressed from vector pPIC3.5K and were fused with a His-tag and green fluorescent protein at the N-terminus. The two plasmids were transformed into P. pastoris with or without PTP1B, resulting in 10 strains. The EGFR-2 and PDGFRß fusion proteins were purified by Ni(2+) affinity chromatography. In the recombinant P. pastoris, the PTKs co-expressed with PTP1B exhibited higher kinase catalytic activity than did those expressing the PTKs alone. The highest activities were achieved by targeting the PTKs and PTP1B into peroxisomes. Therefore, the EGFR-2 and PDGFRß fusion proteins expressed in P. pastoris may be attractive drug screening targets for anticancer therapeutics.

A miniaturized chemical proteomic approach for target profiling of clinical kinase inhibitors in tumor biopsies.

While targeted therapy based on the idea of attenuating the activity of a preselected, therapeutically relevant protein has become one of the major trends in modern cancer therapy, no truly specific targeted drug has been developed and most clinical agents have displayed a degree of polypharmacology. Therefore, the specificity of anticancer therapeutics has emerged as a highly important but severely underestimated issue. Chemical proteomics is a powerful technique combining postgenomic drug-affinity chromatography with high-end mass spectrometry analysis and bioinformatic data processing to assemble a target profile of a desired therapeutic molecule. Due to high demands on the starting material, however, chemical proteomic studies have been mostly limited to cancer cell lines. Herein, we report a down-scaling of the technique to enable the analysis of very low abundance samples, as those obtained from needle biopsies. By a systematic investigation of several important parameters in pull-downs with the multikinase inhibitor bosutinib, the standard experimental protocol was optimized to 100 µg protein input. At this level, more than 30 well-known targets were detected per single pull-down replicate with high reproducibility. Moreover, as presented by the comprehensive target profile obtained from miniaturized pull-downs with another clinical drug, dasatinib, the optimized protocol seems to be extendable to other drugs of interest. Sixty distinct human and murine targets were finally identified for bosutinib and dasatinib in chemical proteomic experiments utilizing core needle biopsy samples from xenotransplants derived from patient tumor tissue. Altogether, the developed methodology proves robust and generic and holds many promises for the field of personalized health care.

Detection of t(2;5)(p23;q35) in anaplastic large-cell lymphoma by long-range nested polymerase chain reaction assay.

Anaplastic large-cell lymphoma (ALCL) is characterized by molecular abnormalities involving ALK gene located at 2p23 which results in an overexpression of ALK. Nine different rearrangements of ALK gene have been reported; the t(2;5)(p23;q35) is the most common. The t(2;5) fuses the NPM gene at 5q35 with the ALK gene. NPM/ALK, as well as ALK activation via other molecular abnormalities, plays an important role in the pathogenesis of ALCL. The lack of tight clustering within the involved NPM and ALK genes precludes analysis using standard PCR methods. We describe a long-range nested PCR assay to detect NPM/ALK gene rearrangements.

A multiple reaction monitoring (MRM) method to detect Bcr-Abl kinase activity in CML using a peptide biosensor.

The protein kinase Bcr-Abl plays a major role in the pathogenesis of chronic myelogenous leukemia (CML), and is the target of the breakthrough drug imatinib (Gleevec™). While most patients respond well to imatinib, approximately 30% never achieve remission or develop resistance within 1-5 years of starting imatinib treatment. Evidence from clinical studies suggests that achieving at least 50% inhibition of a patient's Bcr-Abl kinase activity (relative to their level at diagnosis) is associated with improved patient outcomes, including reduced occurrence of resistance and longer maintenance of remission. Accordingly, sensitive assays for detecting Bcr-Abl kinase activity compatible with small amounts of patient material are desirable as potential companion diagnostics for imatinib. Here we report the detection of Bcr-Abl activity and inhibition by imatinib in the human CML cell line K562 using a cell-penetrating peptide biosensor and multiple reaction monitoring (MRM) on a triple quadrupole mass spectrometer. MRM enabled reproducible, selective detection of the peptide biosensor at fmol levels from aliquots of cell lysate equivalent to ~15,000 cells. This degree of sensitivity will facilitate the miniaturization of the entire assay procedure down to cell numbers approaching 15,000, making it practical for translational applications in patient cells in which the limited amount of available patient material often presents a major challenge.

Evaluation of potential Myt1 kinase inhibitors by TR-FRET based binding assay.

In the human cell cycle, the Myt1 kinase is a crucial regulator of the G2/M transition. Because this membrane-associated kinase is hard to obtain and assay, there is a distinct lack of data so far. Here we report the derivatization of a glycoglycerolipid which was shown previously to be active in a Myt1 activity assay. These compounds were tested in a binding assay together with a set of common kinase inhibitors against a full-length Myt1 expressed in a human cell line. Dasatinib exhibited nanomolar affinity whereas broad coverage inhibitors such as sunitinib and staurosporine derivatives did not show any effect. We also carried out docking studies for the most potent compounds allowing further insights into the inhibitor interaction of this kinase. The glycoglycerolipids showed no significant effects in the binding assay, endorsing the idea of a mechanism of action distant from the active site.

Detection of a rare BCR-ABL tyrosine kinase fusion protein in H929 multiple myeloma cells using immunoprecipitation (IP)-tandem mass spectrometry (MS/MS).

Hypothesis directed proteomics offers higher throughput over global analyses. We show that immunoprecipitation (IP)-tandem mass spectrometry (LC-MS/MS) in H929 multiple myeloma (MM) cancer cells led to the discovery of a rare and unexpected BCR-ABL fusion, informing a therapeutic intervention using imatinib (Gleevec). BCR-ABL is the driving mutation in chronic myeloid leukemia (CML) and is uncommon to other cancers. Three different IP-MS experiments central to cell signaling pathways were sufficient to discover a BCR-ABL fusion in H929 cells: phosphotyrosine (pY) peptide IP, p85 regulatory subunit of phosphoinositide-3-kinase (PI3K) IP, and the GRB2 adaptor IP. The pY peptides inform tyrosine kinase activity, p85 IP informs the activating adaptors and receptor tyrosine kinases (RTKs) involved in AKT activation and GRB2 IP identifies RTKs and adaptors leading to ERK activation. Integration of the bait-prey data from the three separate experiments identified the BCR-ABL protein complex, which was confirmed by biochemistry, cytogenetic methods, and DNA sequencing revealed the e14a2 fusion transcript. The tyrosine phosphatase SHP2 and the GAB2 adaptor protein, important for MAPK signaling, were common to all three IP-MS experiments. The comparative treatment of tyrosine kinase inhibitor (TKI) drugs revealed only imatinib, the standard of care in CML, was inhibitory to BCR-ABL leading to down-regulation of pERK and pS6K and inhibiting cell proliferation. These data suggest a model for directed proteomics from patient tumor samples for selecting the appropriate TKI drug(s) based on IP and LC-MS/MS. The data also suggest that MM patients, in addition to CML patients, may benefit from BCR-ABL diagnostic screening.

Highly efficient purification of protein complexes from mammalian cells using a novel streptavidin-binding peptide and hexahistidine tandem tag system: application to Bruton's tyrosine kinase.

Tandem affinity purification (TAP) is a generic approach for the purification of protein complexes. The key advantage of TAP is the engineering of dual affinity tags that, when attached to the protein of interest, allow purification of the target protein along with its binding partners through two consecutive purification steps. The tandem tag used in the original method consists of two IgG-binding units of protein A from Staphylococcus aureus (ProtA) and the calmodulin-binding peptide (CBP), and it allows for recovery of 20-30% of the bait protein in yeast. When applied to higher eukaryotes, however, this classical TAP tag suffers from low yields. To improve protein recovery in systems other than yeast, we describe herein the development of a three-tag system comprised of CBP, streptavidin-binding peptide (SBP) and hexa-histidine. We illustrate the application of this approach for the purification of human Bruton's tyrosine kinase (Btk), which results in highly efficient binding and elution of bait protein in both purification steps (>50% recovery). Combined with mass spectrometry for protein identification, this TAP strategy facilitated the first nonbiased analysis of Btk interacting proteins. The high efficiency of the SBP-His6 purification allows for efficient recovery of protein complexes formed with a target protein of interest from a small amount of starting material, enhancing the ability to detect low abundance and transient interactions in eukaryotic cell systems.