Advances in biological regulation最新文献

Inositol poly- and pyrophosphates (InsPs and PP-InsPs) are a group of central eukaryotic metabolites and signaling molecules. Due to the diverse cellular functions and widespread diseases InsPs and PP-InsPs are associated with, pharmacological targeting of the kinases involved in their biosynthesis has become a significant research interest in the last decade. In particular, the development of inhibitors for inositol hexakisphosphate kinases (IP6Ks) has leaped forward, while other inositol phosphate kinases have received scant attention.

This review summarizes the efforts undertaken so far for discovering potent and selective inhibitors for this diverse group of small molecule kinases. The benefits of pharmacological inhibition are highlighted, given the multiple kinase-independent functions of inositol phosphate kinases. The distinct structural families of InsP and PP-InsP kinases are presented, and we discuss how compound availability for different inositol phosphate kinase families varies drastically. Lead compound discovery and optimization for the inositol kinases would benefit from detailed structural information on the ATP-binding sites of these kinases, as well as reliable biochemical and cellular read-outs to monitor inositol phosphate kinase activity in complex settings. Efforts to further tune well-established inhibitors, while simultaneously reviving tool compound development for the more neglected kinases from this family are indisputably worthwhile, considering the large potential therapeutic benefits.

Approaches to improve pancreatic cancer therapy are essential as this disease has a very bleak outcome. Approximately 80% of pancreatic cancers are pancreatic ductal adenocarcinomas (PDAC). A key regulatory gene frequently mutated (∼75%) in PDAC is the TP53 tumor suppressor gene which controls the transcription of multiple genes involved in cell cycle progression, apoptosis, cancer progression and other growth regulatory processes. The mouse double minute 2 homolog (MDM2) gene product is a nuclear-localized E3 ubiquitin ligase and negatively regulates the TP53 protein which results in its proteasomal degradation. Various MDM2 inhibitors have been isolated and examined in clinical trials, especially in patients with hematological malignancies. Nutlin-3a is one of the first MDM2 inhibitors isolated. Berberine (BBR) is a natural product found in many fruits and berries and used in traditional medicine for centuries. It has many biological effects, and some are anti-proliferative in nature. BBR may activate the expression of TP53 and inhibit cell cycle progression as well as other events important in cell growth. To understand more about the potential of compounds like BBR and chemical modified BBRs (NAX compounds) to sensitize PDAC cells to MDM2 inhibitors, we introduced either WT-TP53 or the pLXSN empty vector control into two PDAC cell lines, one lacking expression of TP53 (PANC-28) and one with gain-of-function mutant TP53 on both alleles (MIA-PaCa-2). Our results indicate that nutlin-3a was able to increase the sensitivity to BBR and certain NAX compounds. The effects of nutlin-3a were usually more substantial in those cells containing an introduced WT TP53 gene. These results highlight the importance of knowledge of the type of TP53 mutation that is present in cancer patients before the administration of drugs which function by stabilization of the TP53 protein.

The COSMIC database (version 94) lists 576 genes in the Cancer Gene Census which have a defined function as drivers of malignancy (oncogenes) or as tumour suppressors (Tier 1). In addition, there are 147 genes with similar functions, but which are less well characterised (Tier 2). Furthermore, next-generation sequencing projects in the context of precision oncology activities are constantly discovering new ones. Since cancer genes differ from their wild-type precursors in numerous molecular and biochemical properties and exert significant differential effects on downstream processes, simple assays that can uncover oncogenic or anti-oncogenic functionality are desirable and may precede more sophisticated analyses. We describe simple functional assays for PTPN11 (protein-tyrosine phosphatase, non-receptor-type 11)/SHP2 mutants, which are typically found in RASopathies and exhibit potential oncogenic activity. We have also designed a functional test for lysyl oxidase (LOX), a prototypical class II tumour suppressor gene whose loss of function may contribute to neoplastic transformation by RAS oncogenes. Moreover, we applied this test to analyse three co-regulated, RAS-responsive genes for transformation-suppressive activity. The integration of these tests into systems biology studies will contribute to a better understanding of cellular networks in cancer.

Oxysterols are oxygenated cholesterol derivatives and important regulators of cholesterol metabolism, lipid homeostasis, the immune system, and membrane fluidity regulation. Although the detailed mechanism of action of oxysterols remains unclear, activation of some nuclear receptors, such as liver X receptor α (LXRα) and RAR-related orphan receptors, have been believed to be critical for the regulation of various physiological processes in multiple tissues. 27-Hydroxycholesterol (27-OHC) is an endogenous oxysterol, which has an intermediate function in cholesterol catabolism to bile acid synthesis. According to previous studies, however, there are opposing opinions on whether 27-OHC activates human LXR. Recently, several studies have shown that 27-OHC can activate or inhibit the function of estrogen receptors ERα and ERβ in a tissue-specific manner, indicating that the understanding of 27-OHC-mediated biological output is very complicated. This review summarizes the pathophysiological relevance of 27-OHC in various tissues, with a special discussion on their functions in human diseases.

The growth factor Midkine is a heparin-binding cytokine originally discovered during the differentiation process induced by the retinoic acid in embryonal carcinoma cells. Several studies pointed out the key role of this protein in tumour progression and its elevated expression in different malignancies, including pancreatic cancer. New diagnostic and therapeutic tools are urgently required to treat this highly aggressive and incurable disease capable of metastasising, evading diagnosis, and resisting therapy. Serum midkine promises to be a very functional tumour marker and a target for cancer treatment as an elevated concentration of serum midkine is consistently reported in patients with various tumours. Here, we identified high levels of midkine in extracellular vesicles isolated from pancreatic cancer cell lines and showed that it stimulates the growth of pancreatic cancer cells not expressing midkine.

Bisphosphate nucleotidase 2 (BPNT2) is a member of a family of phosphatases that are directly inhibited by lithium, the first-line medication for bipolar disorder. BPNT2 is localized to the Golgi, where it metabolizes the by-products of glycosaminoglycan sulfation reactions. BPNT2-knockout mice exhibit impairments in total-body chondroitin-4-sulfation which lead to abnormal skeletal development (chondrodysplasia). These mice die in the perinatal period, which has previously prevented the investigation of BPNT2 in the adult nervous system. Previous work has demonstrated the importance of chondroitin sulfation in the brain, as chondroitin-4-sulfate is a major component of perineuronal nets (PNNs), a specialized neuronal extracellular matrix which mediates synaptic plasticity and regulates certain behaviors. We hypothesized that the loss of BPNT2 in the nervous system would decrease chondroitin-4-sulfation and PNNs in the brain, which would coincide with behavioral abnormalities. We used Cre-lox breeding to knockout Bpnt2 specifically in the nervous system using Bpnt2 floxed (fl/fl) animals and a Nestin-driven Cre recombinase. These mice are viable into adulthood, and do not display gross physical abnormalities. We identified decreases in total glycosaminoglycan sulfation across selected brain regions, and specifically show decreases in chondroitin-4-sulfation which correspond with increases in chondroitin-6-sulfation. Interestingly, these changes were not correlated with gross alterations in PNNs. We also subjected these mice to a selection of neurobehavioral assessments and did not identify significant behavioral abnormalities. In summary, this work demonstrates that BPNT2, a known target of lithium, is important for glycosaminoglycan sulfation in the brain, suggesting that lithium-mediated inhibition of BPNT2 in the nervous system warrants further investigation.

Calcium-regulated exocytosis is a multi-step process that allows specialized secretory cells to release informative molecules such as neurotransmitters, neuropeptides, and hormones for intercellular communication. The biogenesis of secretory vesicles from the Golgi cisternae is followed by their transport towards the cell periphery and their docking and fusion to the exocytic sites of the plasma membrane allowing release of vesicular content. Subsequent compensatory endocytosis of the protein and lipidic constituents of the vesicles maintains cell homeostasis. Despite the fact that lipids represent the majority of membrane constituents, little is known about their contribution to these processes. Using a combination of electrochemical measurement of single chromaffin cell catecholamine secretion and electron microscopy of roof-top membrane sheets associated with genetic, silencing and pharmacological approaches, we recently reported that diverse phosphatidic acid (PA) species regulates catecholamine release efficiency by controlling granule docking and fusion kinetics. The enzyme phospholipase D1 (PLD1), producing PA from phosphatidylcholine, seems to be the major responsible of these effects in this model. Here, we extended this work using spinning disk confocal microscopy showing that inhibition of PLD activity also reduced the velocity of granules undergoing a directed motion. Furthermore, a dopamine β-hydroxylase (DβH) internalization assay revealed that PA produced by PLD is required for an optimal recovery of vesicular membrane content by compensatory endocytosis. Thus, among numerous roles that have been attributed to PA our work gives core to the key regulatory role in secretion that has been proposed in different cell models. Few leads to explain these multiple functions of PA along the secretory pathway are discussed.

Aberrant signaling of mechanistic target of rapamycin (mTOR aka mammalian target of rapamycin) is shown to be linked to tumorigenesis of numerous malignancies including glioblastoma (GB). mTOR is a serine threonine kinase that functions by forming two multiprotein complexes. These complexes are named mTORC1 and mTORC2 and activate downstream substrates that execute cellular and metabolic functions. This signaling cascade of PI3K/AKT/mTOR is often upregulated due to frequent loss of the tumor suppressor PTEN, a phosphatase that functions antagonistically to PI3K. mTOR regulates cell growth, motility, and metabolism by forming two multiprotein complexes, mTORC1 and mTORC2, which are composed of special binding partners. These complexes are sensitive to distinct stimuli. mTORC1 is sensitive to nutrients and mTORC2 is regulated via PI3K and growth factor signaling. Since rapamycin and its analogue are less effective in treatment of GB, we used novel ATP-competitive dual inhibitors of mTORC1 and mTORC2, namely, Torin1, Torin2, and XL388. Torin2 caused a concentration dependent pharmacodynamic effects on inhibition of phosphorylation of the mTORC1 substrates S6K^Ser235/236 and 4E-BP1^Thr37/46 as well as the mTORC2 substrate AKT^Ser473 resulting in suppression of tumor cell proliferation and migration. Torin1 showed similar effects only at higher doses. Another small molecule compound, XL388 suppressed cell proliferation at a higher dose but failed to inhibit cell migration. Torin1 suppressed phosphorylation of PRAS40^Thr246, however, Torin2 completely abolished it. XL388 treatment inhibited the phosphorylation of PRAS40^Thr246 at higher doses only. These findings underscore the use of novel compounds in treatment of cancer. In addition, formulation of third generation mTOR inhibitor “Rapalink-1” may provide new aspects to target mTOR pathways. Numerous inhibitors are currently being used in clinical trials that are aimed to target activated mTOR pathways.

Phosphoinositide-specific phospholipases C (PLCs) are a class of enzymes involved in several cell activities, such as cell cycle regulation, proliferation, differentiation and cytoskeletal dynamics. Among these enzymes, PLCγ1 is one of the most expressed PLCs in the brain, contributing to a complex network in the developing nervous system. Several studies have shown that PLCγ1 signaling imbalance is linked to several brain disorders, including glioblastoma, the most aggressive brain tumor in adults. Indeed, it has been demonstrated a link between PLCγ1 inhibition and the arrest of glioma cell motility of fetal rat brain aggregates and the impairment of cell invasion abilities following its down-regulation. This study aims to determine the pathological influence of PLCγ1 in glioblastoma, through a translational study which combines in silico data, data from glioblastoma patients' samples and data on engineered cell lines. We found out that PLCγ1 gene expression correlates with the pathological grade of gliomas, and it is higher in fifty patients' glioblastoma tissue samples compared to twenty healthy controls. Moreover, it was demonstrated that PLCγ1 silencing in U87-MG leads to a reduction in cell migration and invasion abilities. The opposite trend was observed following PLCγ1 overexpression, suggesting an interesting possible involvement of PLCγ1 in gliomas' aggressiveness.