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Critical Reviews in Biochemistry and... Apr 2018Protein kinase C (PKC) isozymes belong to a family of Ser/Thr kinases whose activity is governed by reversible release of an autoinhibitory pseudosubstrate. For... (Review)
Review
Protein kinase C (PKC) isozymes belong to a family of Ser/Thr kinases whose activity is governed by reversible release of an autoinhibitory pseudosubstrate. For conventional and novel isozymes, this is effected by binding the lipid second messenger, diacylglycerol, but for atypical PKC isozymes, this is effected by binding protein scaffolds. PKC shot into the limelight following the discovery in the 1980s that the diacylglycerol-sensitive isozymes are "receptors" for the potent tumor-promoting phorbol esters. This set in place a concept that PKC isozymes are oncoproteins. Yet three decades of cancer clinical trials targeting PKC with inhibitors failed and, in some cases, worsened patient outcome. Emerging evidence from cancer-associated mutations and protein expression levels provide a reason: PKC isozymes generally function as tumor suppressors and their activity should be restored, not inhibited, in cancer therapies. And whereas not enough activity is associated with cancer, variants with enhanced activity are associated with degenerative diseases such as Alzheimer's disease. This review describes the tightly controlled mechanisms that ensure PKC activity is perfectly balanced and what happens when these controls are deregulated. PKC isozymes serve as a paradigm for the wisdom of Confucius: "to go beyond is as wrong as to fall short."
Topics: Alzheimer Disease; Animals; Humans; Isoenzymes; Mutation; Neoplasms; Oncogene Proteins; Protein Kinase C; Protein Kinase Inhibitors; Second Messenger Systems; Tumor Suppressor Proteins
PubMed: 29513138
DOI: 10.1080/10409238.2018.1442408 -
Prostaglandins & Other Lipid Mediators Sep 2017The hemiketal (HK) eicosanoids HKE and HKD are the major products resulting from the biosynthetic cross-over of the 5-lipoxygenase and cyclooxygenase-2 pathways. They... (Review)
Review
The hemiketal (HK) eicosanoids HKE and HKD are the major products resulting from the biosynthetic cross-over of the 5-lipoxygenase and cyclooxygenase-2 pathways. They are formed by activated human leukocytes ex vivo, and, therefore, may be involved in regulation of the inflammatory response as autocrine or paracrine mediators. HKE and HKD are not commercially available and, so far, no method for their total chemical synthesis has been reported. The limited availability has impeded the characterization of their biological effects. Here, we describe a method for biomimetic preparation of HKE and HKD by reaction of recombinant human cyclooxygenase-2 with chemically synthesized 5S-HETE. We found that HKE did not induce or inhibit the release of TNFα and IL-1β by human THP-1 monocytes and phorbol ester treatment-derived macrophages.
Topics: Aldehydes; Biomimetics; Chemistry Techniques, Synthetic; Cytokines; Eicosanoids; Humans; Ketones; Macrophages; Monocytes
PubMed: 27596332
DOI: 10.1016/j.prostaglandins.2016.09.001 -
Biomedicine & Pharmacotherapy =... Jul 2023Research on transient receptor potential vanilloid-4 (TRPV4) can provide a promising potential therapeutic target in the development of novel medicines for lung... (Review)
Review
Research on transient receptor potential vanilloid-4 (TRPV4) can provide a promising potential therapeutic target in the development of novel medicines for lung disorders. TRPV4 expresses in lung tissue and plays an important role in the maintenance of respiratory homeostatic function. TRPV4 is upregulated in life-threatening respiratory diseases like pulmonary hypertension, asthma, cystic fibrosis, and chronic obstructive pulmonary diseases. TRPV4 is linked to several proteins that have physiological functions and are sensitive to a wide variety of stimuli, such as mechanical stimulation, changes in temperature, and hypotonicity, and responds to a variety of proteins and lipid mediators, including anandamide (AA), the arachidonic acid metabolite, 5,6-epoxyeicosatrienoic acid (5,6-EET), a plant dimeric diterpenoid called bisandrographolide A (BAA), and the phorbol ester 4-alpha-phorbol-12,13-didecanoate (4α-PDD). This study focused on relevant research evidence of TRPV4 in lung disorders and its agonist and antagonist effects. TRPV4 can be a possible target of discovered molecules that exerts high therapeutic potential in the treatment of respiratory diseases by inhibiting TRPV4.
Topics: Humans; Transient Receptor Potential Channels; TRPV Cation Channels; Phorbol Esters; Hypertension, Pulmonary
PubMed: 37178575
DOI: 10.1016/j.biopha.2023.114861 -
Cells Sep 2023Confocal microscopy and fluorescence staining of cellular structures are commonly used to study neutrophil activation and NETosis. However, they do not reveal the...
Confocal microscopy and fluorescence staining of cellular structures are commonly used to study neutrophil activation and NETosis. However, they do not reveal the specific characteristics of the neutrophil membrane surface, its nanostructure, and morphology. The aim of this study was to reveal the topography and nanosurface characteristics of neutrophils during activation and NETosis using atomic force microscopy (AFM). We showed the main stages of neutrophil activation and NETosis, which include control cell spreading, cell fragment formation, fusion of nuclear segments, membrane disruption, release of neutrophil extracellular traps (NETs), and final cell disintegration. Changes in neutrophil membrane nanosurface parameters during activation and NETosis were quantified. It was shown that with increasing activation time there was a decrease in the spectral intensity of the spatial periods. Exposure to the activator A23187 resulted in an increase in the number and average size of cell fragments over time. Exposure to the activators A23187 and PMA (phorbol 12-myristate 13-acetate) caused the same pattern of cell transformation from spherical cells with segmented nuclei to disrupted cells with NET release. A23187 induced NETosis earlier than PMA, but PMA resulted in more cells with NETosis at the end of the specified time interval (180 min). In our study, we used AFM as the main research tool. Confocal laser-scanning microscopy (CLSM) images are provided for identification and detailed analysis of the phenomena studied. In this way, we exploited the advantages of both techniques.
Topics: Neutrophils; Calcimycin; Microscopy, Atomic Force; Extracellular Traps; Cell Nucleus; Tetradecanoylphorbol Acetate
PubMed: 37681931
DOI: 10.3390/cells12172199 -
Frontiers in Immunology 2022Neutrophil extracellular traps (NETs)-as double-edged swords of innate immunity-are involved in numerous processes such as infection, inflammation and tissue repair....
BACKGROUND
Neutrophil extracellular traps (NETs)-as double-edged swords of innate immunity-are involved in numerous processes such as infection, inflammation and tissue repair. Research on neutrophil granulocytes is limited because of their short lifetime of only a few hours. Several attempts have been made to prolong the half-life of neutrophils using cytokines and bacterial products and have shown promising results. These long-term surviving neutrophils are reported to maintain phagocytic activity and cytokine release; however, little is known regarding their capability to release NETs.
METHODS
We analysed the prolongation of neutrophil survival under various culture conditions using granulocyte colony-stimulating factor (G-CSF), lipopolysaccharide (LPS) or tumour necrosis factor alpha (TNF-α) by flow cytometry and a viability assay. Additionally, we assessed NET formation following stimulation with phorbol 12-myristate 13-acetate (PMA) by immunofluorescence staining, myeloperoxidase (MPO)-DNA sandwich-ELISA and fluorometric assays for cell-free DNA (cfDNA), neutrophil elastase (NE) and myeloperoxidase (MPO).
RESULTS
Untreated neutrophils could form NETs after stimulation with PMA for up to 24 h. Incubation with LPS extended their ability to form NETs for up to 48 h. At 48 h, NET release of neutrophils cultured with LPS was significantly higher compared to that of untreated cells; however, no significantly different enzymatic activity of NE and MPO was observed. Similarly, incubation with G-CSF resulted in significantly higher NET release at 48 h compared to untreated cells. Furthermore, NETs showed significantly higher enzymatic activity of NE and MPO after incubation with G-CSF. Lastly, incubation with TNF-α had no influence on NET release compared to untreated cells although survival counts were altered by TNF-α.
CONCLUSIONS
G-CSF, LPS or TNF-α each at low concentrations lead to prolonged survival of cultured neutrophils, resulting in considerable differences in NET formation and composition. These results provide new information for the use of neutrophils in long-term experiments for NET formation and provide novel insights for neutrophil behaviour under inflammatory conditions.
Topics: Cytokines; Granulocyte Colony-Stimulating Factor; Lipopolysaccharides; Neutrophils; Peroxidase; Tetradecanoylphorbol Acetate; Tumor Necrosis Factor-alpha
PubMed: 35242132
DOI: 10.3389/fimmu.2022.815412 -
Journal of Cancer 2021Protein kinase D3 (PRKD3), a serine/threonine kinase, belongs to protein kinase D family, which contains three members: PRKD1, PRKD2, and PRKD3. PRKD3 is activated by... (Review)
Review
Protein kinase D3 (PRKD3), a serine/threonine kinase, belongs to protein kinase D family, which contains three members: PRKD1, PRKD2, and PRKD3. PRKD3 is activated by many stimuli including phorbol esters, and G-protein-coupled receptor agonists. PRKD3 promotes cancer cell proliferation, growth, migration, and invasion in various tumor types including colorectal, gastric, hepatic, prostate, and breast cancer. Accumulating data supports that PRKD3 is a promising therapeutic target for treatment of cancer. This review discusses the functions and mechanisms of PRKD3 in promoting tumorigenesis and tumor progression of various tumor types as well as the latest developments of small-molecule inhibitors selection for PRKD/PRKD3.
PubMed: 33403031
DOI: 10.7150/jca.50899 -
Journal of the American Chemical Society Nov 2020Self-assembly of amphiphilic peptide-based building blocks gives rise to a plethora of interesting nanostructures such as ribbons, fibers, and tubes. However, it remains...
Self-assembly of amphiphilic peptide-based building blocks gives rise to a plethora of interesting nanostructures such as ribbons, fibers, and tubes. However, it remains a great challenge to employ peptide self-assembly to directly produce nanostructures with lower symmetry than these highly symmetric motifs. We report here our discovery that persistent and regular crescent nanostructures with a diameter of 28 ± 3 nm formed from a series of tetrapeptides with the general structure AdKKEX (Ad = adamantyl group, K = lysine residue functionalized with an -aroylthiooxime (SATO) group, E = glutamic acid residue, and X = variable amino acid residue). In the presence of cysteine, the biological signaling gas hydrogen sulfide (HS) was released from the SATO units of the crescent nanostructures, termed peptide-HS donor conjugates (PHDCs), reducing levels of reactive oxygen species (ROS) in macrophage cells. Additional studies showed that the crescent nanostructures alleviated cytotoxicity induced by phorbol 12-myristate-13-acetate more effectively than common HS donors and a PHDC of a similar chemical structure, AdKKE, that formed short nanoworms instead of nanocrescents. Cell internalization studies indicated that nanocrescent-forming PHDCs were more effective in reducing ROS levels in macrophages because they entered into and remained in cells better than nanoworms, highlighting how nanostructure morphology can affect bioactivity in drug delivery.
Topics: Animals; Cell Survival; Hydrogen Sulfide; Macrophages; Mice; Nanostructures; Oligopeptides; RAW 264.7 Cells; Reactive Oxygen Species; Tetradecanoylphorbol Acetate
PubMed: 33186019
DOI: 10.1021/jacs.0c09399 -
Journal of Natural Products Aug 2022The kernels of the Australian blushwood tree () are the source of the veterinary anticancer drug tigilanol tiglate (, Stelfonta) and contain a concentration of phorboids... (Review)
Review
The kernels of the Australian blushwood tree () are the source of the veterinary anticancer drug tigilanol tiglate (, Stelfonta) and contain a concentration of phorboids significantly higher than croton oil, the only abundant source of these compounds previously known. The oily matrix of the blushwood kernels is composed of free fatty acids and not by glycerides as found in croton oil. By active partitioning, it was therefore possible to recover and characterize for the first time a cryptic tigliane fraction, that is, the diterpenoid fraction that, because of its lipophilicity, could not be obtained by solvent partition of crude extracts. The cryptic tigliane fraction accounted for ca. 30% of the tigliane kernel titer and was quantified by H NMR spectroscopy and profiled by HPLC-MS. Long-chain (linoleates and/or oleates) 20-acyl derivatives of the epoxytigliane diesters tigilanol tiglate (EBC-46, ), EBC-47 (), EBC-59 (), EBC-83 (), and EBC-177 () were identified. By chemoselective acylation of EBC-46 () and EBC-177 () the natural triesters and and a selection of analogues were prepared to assist identification of the natural compounds. The presence of a free C-20 hydroxy group is a critical requirement for PKC activation by phorbol esters. The unexpected activity of 20-linoleoyl triester in a cytotoxicity assay based on PKC activation was found to be related mainly to its hydrolysis to tigilanol tiglate () under the prolonged conditions of the assay, while other esters were inactive. Significant differences between the esterification profile of the epoxytigliane di- and triesters exist in , suggesting a precise, yet elusive, blueprint of acyl decoration for the tigliane polyol 5-hydroxyepoxyphorbol.
Topics: Australia; Croton Oil; Euphorbiaceae; Phorbols; Trees
PubMed: 35973043
DOI: 10.1021/acs.jnatprod.2c00226 -
The Journal of Biological Chemistry May 2019G protein-coupled receptor (GPCR) signaling is regulated by members of the protein kinase C (PKC) and GPCR kinase (GRK) families, although the relative contribution of...
G protein-coupled receptor (GPCR) signaling is regulated by members of the protein kinase C (PKC) and GPCR kinase (GRK) families, although the relative contribution of each to GPCR function varies among specific GPCRs. The CC motif receptor 4 (CXCR4) is a member of the GPCR superfamily that binds the CC motif chemokine ligand 12 (CXCL12), initiating signaling that is subsequently terminated in part by internalization and lysosomal degradation of CXCR4. The purpose of this study is to define the relative contribution of PKC and GRK to CXCR4 signaling attenuation by studying their effects on CXCR4 lysosomal trafficking and degradation. Our results demonstrate that direct activation of PKC via the phorbol ester phorbol 12-myristate 13-acetate (PMA) mimics CXCL12-mediated desensitization, internalization, ubiquitination, and lysosomal trafficking of CXCR4. In agreement, heterologous activation of PKC by stimulating the chemokine receptor CXCR5 with its ligand, CXCL13, also mimics CXCL12-mediated desensitization, internalization, ubiquitination, and lysosomal degradation of CXCR4. Similar to CXCL12, PMA promotes PKC-dependent phosphorylation of serine residues within CXCR4 C-tail that are required for binding and ubiquitination by the E3 ubiquitin ligase AIP4 (atrophin-interacting protein 4). However, inhibition of PKC activity does not alter CXCL12-mediated ubiquitination and degradation of CXCR4, suggesting that other kinases are also required. Accordingly, siRNA-mediated depletion of GRK6 results in decreased degradation and ubiquitination of CXCR4. Overall, these results suggest that PKC and GRK6 contribute to unique aspects of CXCR4 phosphorylation and lysosomal degradation to ensure proper signal propagation and termination.
Topics: Chemokine CXCL12; Chemokine CXCL13; Enzyme Activation; G-Protein-Coupled Receptor Kinases; HEK293 Cells; HeLa Cells; Humans; Lysosomes; Protein Kinase C; Protein Transport; Proteolysis; Receptors, CXCR4; Repressor Proteins; Signal Transduction; Tetradecanoylphorbol Acetate; Ubiquitin-Protein Ligases; Ubiquitination
PubMed: 30936203
DOI: 10.1074/jbc.RA118.005991 -
Seminars in Cancer Biology Feb 2018Protein kinase C (PKC) has historically been considered an oncoprotein. This stems in large part from the discovery in the early 1980s that PKC is directly activated by... (Review)
Review
Protein kinase C (PKC) has historically been considered an oncoprotein. This stems in large part from the discovery in the early 1980s that PKC is directly activated by tumor-promoting phorbol esters. Yet three decades of clinical trials using PKC inhibitors in cancer therapies not only failed, but in some cases worsened patient outcome. Why has targeting PKC in cancer eluded successful therapies? Recent studies looking at the disease for insight provide an explanation: cancer-associated mutations in PKC are generally loss-of-function (LOF), supporting an unexpected function as tumor suppressors. And, contrasting with LOF mutations in cancer, germline mutations that enhance the activity of some PKC isozymes are associated with degenerative diseases such as Alzheimer's disease. This review provides a background on the diverse mechanisms that ensure PKC is only active when, where, and for the appropriate duration needed and summarizes recent findings converging on a paradigm reversal: PKC family members generally function by suppressing, rather than promoting, survival signaling.
Topics: Enzyme Activation; Genes, Tumor Suppressor; Humans; Isoenzymes; Mutation; Neoplasms; Phorbol Esters; Protein Kinase C; Signal Transduction
PubMed: 28476658
DOI: 10.1016/j.semcancer.2017.04.017