The chemically synthesized ageladine A-derivative LysoGlow84 stains lysosomes in viable mammalian brain cells and specific structures in the marine flatworm Macrostomum lignano.

Based on the chemical structure and the known chemical synthesis of the marine sponge alkaloid ageladine A, we synthesized the ageladine A-derivative 4-(naphthalene-2-yl)-1H-imidazo[4,5-c]pyridine trifluoroacetate (LysoGlow84). The two-step synthesis started with the Pictet-Spengler reaction of histamine and naphthalene-2-carbaldehyde to a tetrahydropyridine intermediate, which was dehydrogenated with activated manganese (IV) oxide to LysoGlow84. Structure and purity of the synthesized LysoGlow84 were confirmed by NMR spectroscopy and mass spectrometry. The fluorescence intensity emitted by LysoGlow84 depended strongly on the pH of the solvent with highest fluorescence intensity recorded at pH 4. The fluorescence maximum (at 315 nm excitation) was observed at 440 nm. Biocompatibility of LysoGlow84 was investigated using cultured rat brain astrocytes and the marine flatworm Macrostomum lignano. Exposure of the astrocytes for up to 6 h to micromolar concentrations of LysoGlow84 did not compromise cell viability, as demonstrated by several viability assays, but revealed a promising property of this compound for staining of cellular vesicles. Conventional fluorescence microscopy as well as confocal scanning microscopy of LysoGlow84-treated astrocytes revealed co-localization of LysoGlow84 fluorescence with that of LysoTracker® Red DND-99. LysoGlow84 stained unclear structures in Macrostomum lignano, which were identified as lysosomes by co-staining with LysoTracker. Strong fluorescence staining by LysoGlow84 was further observed around the worms' anterior gut and the female genital pore which were not counterstained by LysoTracker Red. Thus, LysoGlow84 is a new promising dye that stains lysosomes and other acidic compartments in cultured cells and in worms.

PP2A-B55: substrates and regulators in the control of cellular functions.

PP2A is a major serine/threonine phosphatase class involved in the regulation of cell signaling through the removal of protein phosphorylation. This class of phosphatases is comprised of different heterotrimeric complexes displaying distinct substrate specificities. The present review will focus on one specific heterocomplex, the phosphatase PP2A-B55. Herein, we will report the direct substrates of this phosphatase identified to date, and its impact on different cell signaling cascades. We will additionally describe its negative regulation by its inhibitors Arpp19 and ENSA and their upstream kinase Greatwall. Finally, we will describe the essential molecular features defining PP2A-B55 substrate specificity that confer the correct temporal pattern of substrate dephosphorylation. The main objective of this review is to provide the reader with a unique source compiling all the knowledge of this particular holoenzyme that has evolved as a key enzyme for cell homeostasis and cancer development.

Inhibition of PHLPP1/2 phosphatases rescues pancreatic ß-cells in diabetes.

Pancreatic ß-cell failure is the key pathogenic element of the complex metabolic deterioration in type 2 diabetes (T2D); its underlying pathomechanism is still elusive. Here, we identify pleckstrin homology domain leucine-rich repeat protein phosphatases 1 and 2 (PHLPP1/2) as phosphatases whose upregulation leads to ß-cell failure in diabetes. PHLPP levels are highly elevated in metabolically stressed human and rodent diabetic ß-cells. Sustained hyper-activation of mechanistic target of rapamycin complex 1 (mTORC1) is the primary mechanism of the PHLPP upregulation linking chronic metabolic stress to ultimate ß-cell death. PHLPPs directly dephosphorylate and regulate activities of ß-cell survival-dependent kinases AKT and MST1, constituting a regulatory triangle loop to control ß-cell apoptosis. Genetic inhibition of PHLPPs markedly improves ß-cell survival and function in experimental models of diabetes in vitro, in vivo, and in primary human T2D islets. Our study presents PHLPPs as targets for functional regenerative therapy of pancreatic ß cells in diabetes.

PP2A-B55 Holoenzyme Regulation and Cancer.

Protein phosphorylation is a post-translational modification essential for the control of the activity of most enzymes in the cell. This protein modification results from a fine-tuned balance between kinases and phosphatases. PP2A is one of the major serine/threonine phosphatases that is involved in the control of a myriad of different signaling cascades. This enzyme, often misregulated in cancer, is considered a tumor suppressor. In this review, we will focus on PP2A-B55, a particular holoenzyme of the family of the PP2A phosphatases whose specific role in cancer development and progression has only recently been highlighted. The discovery of the Greatwall (Gwl)/Arpp19-ENSA cascade, a new pathway specifically controlling PP2A-B55 activity, has been shown to be frequently altered in cancer. Herein, we will review the current knowledge about the mechanisms controlling the formation and the regulation of the activity of this phosphatase and its misregulation in cancer.

Neratinib protects pancreatic beta cells in diabetes.

The loss of functional insulin-producing ß-cells is a hallmark of diabetes. Mammalian sterile 20-like kinase 1 (MST1) is a key regulator of pancreatic ß-cell death and dysfunction; its deficiency restores functional ß-cells and normoglycemia. The identification of MST1 inhibitors represents a promising approach for a ß-cell-protective diabetes therapy. Here, we identify neratinib, an FDA-approved drug targeting HER2/EGFR dual kinases, as a potent MST1 inhibitor, which improves ß-cell survival under multiple diabetogenic conditions in human islets and INS-1E cells. In a pre-clinical study, neratinib attenuates hyperglycemia and improves ß-cell function, survival and ß-cell mass in type 1 (streptozotocin) and type 2 (obese Leprdb/db) diabetic mouse models. In summary, neratinib is a previously unrecognized inhibitor of MST1 and represents a potential ß-cell-protective drug with proof-of-concept in vitro in human islets and in vivo in rodent models of both type 1 and type 2 diabetes.

UBASH3B-mediated silencing of the mitotic checkpoint: Therapeutic perspectives in cancer.

Defects in mitosis can lead to aneuploidy, which is a common feature of human cancers. Spindle Assembly Checkpoint (SAC) controls fidelity of chromosome segregation in mitosis to prevent aneuploidy. The ubiquitin receptor protein Ubiquitin Associated and SH3 Domain Containing B (UBASH3B) was recently found to control SAC silencing and faithful chromosome segregation by relocalizing Aurora B kinase to the mitotic microtubules. Accordingly, loss and gain of function of UBASH3B have strong effects on mitotic progression. Downregulation of UBASH3B prevents SAC satisfaction leading to inhibition of chromosome segregation, mitotic arrest, and cell death. In contrast, increased cellular levels of UBASH3B trigger premature and uncontrolled chromosome segregation. Interestingly, elevated levels of UBASH3B were found in aggressive tumors. Therefore, we raised the question whether the oncogenic potential of UBASH3B is linked to its role in chromosome segregation. Here we show that in cancer cells expressing high levels of UBASH3B and SAC proteins, downregulation of UBASH3B, can further potentiate SAC response inducing mitotic arrest and cell death. Moreover, data mining approaches identified a correlation between mRNA levels of UBASH3B and SAC components in a set of primary patient tumors including kidney and liver carcinomas. Thus, inhibition of UBASH3B may offer an attractive therapeutic perspective for cancers.

Proproliferative and antiapoptotic action of exogenously introduced YAP in pancreatic ß cells.

Loss of functional pancreatic ß cells is a hallmark of both type 1 and 2 diabetes. Identifying the pathways that promote ß cell proliferation and/or block ß cell apoptosis is a potential strategy for diabetes therapy. The transcriptional coactivator Yes-associated protein (YAP), a major downstream effector of the Hippo signaling pathway, is a key regulator of organ size and tissue homeostasis by modulating cell proliferation and apoptosis. YAP is not expressed in mature primary human and mouse ß cells. We aimed to identify whether reexpression of a constitutively active form of YAP promotes ß cell proliferation/survival. Overexpression of YAP remarkably induced ß cell proliferation in isolated human islets, while ß cell function and functional identity genes were fully preserved. The transcription factor forkhead box M1 (FOXM1) was upregulated upon YAP overexpression and necessary for YAP-dependent ß cell proliferation. YAP overexpression protected ß cells from apoptosis triggered by multiple diabetic conditions. The small redox proteins thioredoxin-1 and thioredoxin-2 (Trx1/2) were upregulated by YAP; disruption of the Trx system revealed that Trx1/2 was required for the antiapoptotic action of YAP in insulin-producing ß cells. Our data show the robust proproliferative and antiapoptotic function of YAP in pancreatic ß cells. YAP reconstitution may represent a disease-modifying approach to restore a functional ß cell mass in diabetes.