16 research outputs found
Therapeutic Targeting the Cell Division Cycle 25 (CDC25) Phosphatases in Human Acute Myeloid Leukemia — The Possibility to Target Several Kinases through Inhibition of the Various CDC25 Isoforms
No full textThe cell division cycle 25 (CDC25) phosphatases include CDC25A, CDC25B and CDC25C. These three molecules are important regulators of several steps in the cell cycle, including the activation of various cyclin-dependent kinases (CDKs). CDC25s seem to have a role in the development of several human malignancies, including acute myeloid leukemia (AML); and CDC25 inhibition is therefore considered as a possible anticancer strategy. Firstly, upregulation of CDC25A can enhance cell proliferation and the expression seems to be controlled through PI3K-Akt-mTOR signaling, a pathway possibly mediating chemoresistance in human AML. Loss of CDC25A is also important for the cell cycle arrest caused by differentiation induction of malignant hematopoietic cells. Secondly, high CDC25B expression is associated with resistance against the antiproliferative effect of PI3K-Akt-mTOR inhibitors in primary human AML cells, and inhibition of this isoform seems to reduce AML cell line proliferation through effects on NFκB and p300. Finally, CDC25C seems important for the phenotype of AML cells at least for a subset of patients. Many of the identified CDC25 inhibitors show cross-reactivity among the three CDC25 isoforms. Thus, by using such cross-reactive inhibitors it may become possible to inhibit several molecular events in the regulation of cell cycle progression and even cytoplasmic signaling, including activation of several CDKs, through the use of a single drug. Such combined strategies will probably be an advantage in human cancer treatment
Switching of OAS1 splicing isoforms overcomes SNP-derived vulnerability to SARS-CoV-2 infection
No full textRNAスプライシング制御によるCOVID-19重症化リスク低減 --遺伝的脆弱性を緩和する薬剤候補の同定-- .京都大学プレスリリース. 2025-03-04.Background: The SARS-CoV-2 pandemic provided important insights into the relationship between infectious diseases and the human genome. A genomic region encoding the 2′-5′-oligoadenylate synthetase (OAS) family proteins that sense viral genomic RNAs and trigger an antiviral response contains single nucleotide polymorphisms (SNPs) associated with SARS-CoV-2 infection susceptibility. A high-risk SNP identified at the splice acceptor site of OAS1 exon 6 --a terminal exon-- alters the proportion of various splicing isoforms of OAS1 and its activity. However, the actual causality of this SNP or splicing to infection susceptibility remains unknown. Results: In this study, it was found that serine–arginine-rich splicing factor 6 (SRSF6) binds to the splice donor site of the human OAS1 exon 5. SRSF6 determines the selected alternative terminal exon when the risk allele disrupts the splice acceptor site. Subsequently, an inhibitor for CDC-like kinase was rationally selected as a candidate splicing modulator. RNA-Seq and RT-PCR analyses revealed that this inhibitor can induce splice switching of OAS1 mRNAs in the human lung adenocarcinoma cell line Calu-3. Under the inhibitor treatment, the cells exhibited reduced SARS-CoV-2 infection rates. Meanwhile, the colonic epithelial cell line Caco-2 expressed non-risk type OAS1 mRNA isoforms that did not undergo splice-switching or demonstrate altered SARS-CoV-2 sensitivity following treatment with the inhibitor. Conclusions: These results indicate that a high-risk SNP in OAS1 influences cell susceptibility to SARS-CoV-2 infection by inducing splice-switching at its terminal exon. Additionally, chemical splicing modifiers may prove beneficial in overcoming this genomic vulnerability
Myofilament Phosphorylation in Stem Cell Treated Diastolic Heart Failure
No full textRATIONALE: Phosphorylation of sarcomeric proteins has been implicated in heart failure with preserved ejection fraction (HFpEF); such changes may contribute to diastolic dysfunction by altering contractility, cardiac stiffness, Ca(2+)-sensitivity and mechanosensing. Treatment with cardiosphere-derived cells (CDCs) restores normal diastolic function, attenuates fibrosis and inflammation, and improves survival in a rat HFpEF model. OBJECTIVE: Phosphorylation changes that underlie HFpEF and those reversed by CDC therapy, with a focus on the sarcomeric subproteome were analyzed. METHODS AND RESULTS: Dahl salt-sensitive rats fed a high-salt diet, with echocardiographically-verified diastolic dysfunction, were randomly assigned to either intracoronary CDCs or placebo. Dahl salt-sensitive rats receiving low salt diet served as controls. Protein, and phosphorylated Ser, Thr and Tyr residues from left ventricular tissue, were quantified by mass spectrometry. HFpEF hearts exhibited extensive hyperphosphorylation with 98% of the 529 significantly changed phospho-sites increased compared to control. Of those 39% were located within the sarcomeric subproteome, with a large group of proteins located or associated with the Z-disk. CDC treatment partially reverted the hyperphosphorylation, with 85% of the significantly altered 76 residues hypophosphorylated. Bioinformatic upstream analysis of the differentially phosphorylated protein residues revealed PKC as the dominant putative regulatory kinase. PKC isoform analysis indicated increases in PKC α, β and δ concentration, whereas CDC treatment led to a reversion of PKCβ. Use of PKC isoform specific inhibition and overexpression of various PKC isoforms strongly suggests PKCβ is the dominant kinase involved in hyperphosphorylation in HFpEF and is altered with CDC treatment. CONCLUSION: Increased protein phosphorylation at the Z-disk is associated with diastolic dysfunction, with PKC isoforms driving most quantified phosphorylation changes. Because CDCs reverse the key abnormalities in HFpEF and selectively reverse PKCβ upregulation, PKCβ merits being classified as a potential therapeutic target in HFpEF, a disease notoriously refractory to medical intervention
Characterization of the down-regulation of SUMO E2 Ubc9 by cholesterol-dependent cytolysins
No full textPh.D.Cholesterol-dependent cytolysins (CDCs) belong to one of the families of pore-forming toxins that highly associated with bacterial pathogenesis. Membrane disruption of the infected cells is thought to be the major function of CDCs. Interestingly, increasing number of studies have shown that CDCs can also influence diverse post-translational modifications in infected cells, including phosphorlyation, acetylation and SUMOylation. It has been shown that various CDCs including listeriolysin O (LLO), perfringolysin O (PFO) and pneumolysin (PLY) triggered a global suppression of SUMOylation by degrading SUMO E2 conjugating enzyme Ubc9 through a proteasome-independent pathway. This study aims to understand the underlying mechanism of LLO-induced Ubc9 degradation and to investigate whether other CDCs exploit the same strategy in modulating SUMO modification.Recombinant LLO, PLY, SLY and SLO proteins were cloned and purified. The hemolytic activities of all four CDCs were characterized to be at nano molar concentration range. In line with previous study, Western analysis showed that incubation of purified LLO and PLY with HeLa cells led to a decrease of Ubc9 level and overall SUMOylation level. Besides, SLY and SLO exerted does-dependent and proteasome-independent effects on Ubc9, resembling those of LLO and PLY. To map the structural element of Ubc9 essential for the CDC-induced Ubc9 degradation, various Ubc9 mutants defective in SUMO conjugation and phosphorylation were constructed. However, neither SUMO-conjugation nor phosphorylation of Ubc9 was found to have any influence on its degradation. Surprisingly, Ubc9 were degraded more severely in the presence of phosphatase inhibitors, Na3VO4 and NaF, which targets Tyr- and Ser-Thr- phosphotases, respectively. Since both Na3VO4 and NaF can also inhibit cellular ATPase activity, by testing various kinds of ATPase inhibitors, we found that 4-HNE treatment, which inhibiting Na+/K+-ATPase, showed similar effect on Ubc9 level as well as Na3VO4 and NaF treatments, indicating that a yet-to-be identified Na+/K+-ATPase is involved in CDC-induced Ubc9 degradation. Interesting, our result showed that K+ efflux is critical for the down-regulation of Ubc9. However, opposite effect of K+ efflux on Ubc9 degradation was found in SLY.On the other hand, Ca2+-dependent cell blebbing was detected in response to LLO pores. By combining the results from live cell imaging and Western analysis, we surprisingly found that LLO induced release of Ubc9 to extracellular environment, instead of degradation. Interestingly, the three SUMO isoforms but not some other nuclear and cytosolic proteins, can be detected in extracellular space, indicating that LLO triggers the release of Ubc9 and SUMO specifically.In conclusion, this study provides insights to the Ubc9 down-regulation induced by CDCs. Our results reveal for the first time that 1) K+ efflux regulates Ubc9 translocation, and 2) CDCs from different bacterial species may exploit different strategies to benefit their infection. Our findings will offer more understanding about the pathogenesis in CDC-baring pathogens and the development of potential anti-bacterial agents in long term.許多病原體會以宿主細胞的翻譯後修飾為目標，進而感染宿主細胞。膽固醇依賴的溶細胞素（CDC）是與細菌致病性高度相關的一類成孔毒素。它們在細胞膜上形成溶膜孔被認為是CDC的主要功能。然而，越來越多的研究表明，CDC還可以影響宿主細胞中的多種翻譯後修飾過程，其中包括磷酸化修飾、乙醯化修飾和類泛素化修飾。研究顯示，包括LLO,PFO和PLY在內的多種CDC通過降解SUMO E2結合酶Ubc9的非泛素蛋白酶降解途徑，從而抑制宿主細胞內的類泛素化過程。本研究旨在瞭解LLO誘導Ubc9降解的分子機制，以及其他CDC是否共用相同的宿主細胞侵染策略。我們對重組LLO,PLYSLY和SLO蛋白進行克隆並純化，並發現它們都能在納米摩爾濃度水準上具有溶血活性。免疫印跡試驗分析表明，純化的重組LLO和PLY與HeLa細胞進行孵化會導致細胞內Ubc9分子水準及類泛素化水準的降低。此實驗結果與Ribet等（2010）的研究相符。同時，SLY和SLO對Ubc9的降解作用與LLO和PLY的相同，同樣與蛋白劑量正相關及與泛素蛋白酶降解系統無關。為了研究Ubc9結構其降解的關係，我們構建了一系列與類泛素分子SUMO之間有相互作用及與Ubc9自身的磷酸化修飾無關的Ubc9突變體。然而，以上作用位點均與Ubc9的降解無關。有趣的是，磷酸化酶抑制劑Na3VO4和NaF會加速Ubc9的降解。Na3VO4抑制酪氨酸位點的磷酸化酶，而NaF抑制絲氨酸及蘇氨酸位點的磷酸化酶。同時，Na3VO4和NaF都能抑制細胞內的ATP酶活性。通過試驗不同種類的ATP酶抑制劑，我們發現4-HNE同樣可以促進Ubc9的降解，而4-HNE是一種抑制鉀鈉泵活性的抑制劑。巧合的是，我們發現K+外流參與到LLO引起的Ubc9降解過程中。另一方面，我們觀察到宿主細胞能通過細胞膜起泡的方式來抵禦LLO的溶孔入侵。結合活細胞成像和免疫印跡試驗分析，我們發現LLO能使Ubc9發生細胞內轉移並最後釋放到細胞外，而並不是Ubc9降解。有趣的是，經過LLO處理後，三種SUMO亞型都可以在細胞外檢測得到，然而其他測試蛋白卻不能被檢測，意味著LLO引起的Ubc9和SUMO的釋放具有特異性。本研究通過對CDC誘導Ubc9轉移的分子機制進行研究，第一次闡述了1）K+外流可以調控Ubc9的轉移，和2）不同來源的CDC在進行侵染宿主細胞時表現出不同的侵染策略。本研究的研究結果將為以後預防和控制具有CDC的致病菌提供更多的實驗依據。Li, Jiexin.Thesis Ph.D. Chinese University of Hong Kong 2017.Includes bibliographical references (leaves 152-164).Abstracts also in Chinese.Title from PDF title page (viewed on 21, August, 2019)
Yeast-based strategy for identification of 11β-HSD1 inhibitors
No full textGlucocorticoids are hormones with a vital role in regulation of metabolic and defence responses. Their metabolism plays an important role in the pathogenesis of obesity, a major risk factor for metabolic syndrome, a collection of disorders such as insulin resistance, dyslipidemia and hypertension.
At intracellular level the production of active glucocorticoids is regulated by two hydroxysteroid dehydrogenase enzyme isoforms 11β-HSD1 and 11β-HSD2, respectively. 11β-HSD2 is predominantly expressed in mineralocorticoid target tissues and acts as oxidase catalysing conversion of cortisol into cortisone. The isoform 11β-HSD1, expressed in a wide array of tissues and with highest levels in the liver, acts mainly as a reductase converting the cortisone into the biologically active hormone cortisol, in a NADPH dependent manner. Due to its implication in the metabolism of glucocorticoids, 11β-HSD1 has become a primary target for the treatment of metabolic syndrome.
In the last years many strategies have been developed for the screening of molecules with inhibitory effects against this target enzyme. All known approaches present unique features and are suitable for specific screening models. Nevertheless, due to the great therapeutic and economical interest around the treatment of metabolic syndrome, many research groups are constantly focused on the development of new and more successful strategies for identification of hits with improved pharmaceutical properties.
In this study an innovative synthetic biology platform for synthesis and screening of 11β-HSD1 inhibitors has been conceived and investigated. This strategy is based on the production of diverse chemical scaffolds in the yeast Saccharomyces cerevisiae, in which the molecules are directly screened through intracellular functional assays.
Providing all necessary genetic information, the biosynthetic pathway of plant triterpenoids, a big class of natural compounds with many beneficial effects on human health, was reconstructed in the baker’s yeast. Moreover, by applying DNA family shuffling methods, a library of chimeric triterpenoid synthase cDNAs was constructed. The chimeric sequences, potential carriers of new enzymatic functions, were assembled together with the wild-type molecules in expressible yeast artificial chromosomes (eYACs).
Furthermore, the construction of two yeast assays strains functionally co-expressing the murine glucocorticoid receptor (GR) and the human 11β-HSD1 enzyme is presented. In both developed strains, with different mechanisms, the reductase activity of the enzyme 11β-HSD1 on cortisone is connected with an easily detectable fluorescent signal. Cells that produce compounds with inhibitory activity against 11β-HSD1 can be identified through changes in fluorescence and isolated to further investigate the active molecules. The developed yeast-assays were validated using carbenoxolone, a known 11β-HSD1 inhibitor, and may represent useful tools for a first quick and easy screening of large number of membrane permeable putative inhibitory compounds
A novel strategy to characterize the pattern of β-lactam antibiotic-induced drug resistance in Acinetobacter baumannii
No full textAbstract Carbapenem-resistant Acinetobacter baumannii (CRAb) is an urgent public health threat, according to the CDC. This pathogen has few treatment options and causes severe nosocomial infections with > 50% fatality rate. Although previous studies have examined the proteome of CRAb, there have been no focused analyses of dynamic changes to β-lactamase expression that may occur due to drug exposure. Here, we present our initial proteomic study of variation in β-lactamase expression that occurs in CRAb with different β-lactam antibiotics. Briefly, drug resistance to Ab (ATCC 19606) was induced by the administration of various classes of β-lactam antibiotics, and the cell-free supernatant was isolated, concentrated, separated by SDS-PAGE, digested with trypsin, and identified by label-free LC–MS-based quantitative proteomics. Thirteen proteins were identified and evaluated using a 1789 sequence database of Ab β-lactamases from UniProt, the majority of which were Class C β-lactamases (≥ 80%). Importantly, different antibiotics, even those of the same class (e.g. penicillin and amoxicillin), induced non-equivalent responses comprising various isoforms of Class C and D serine-β-lactamases, resulting in unique resistomes. These results open the door to a new approach of analyzing and studying the problem of multi-drug resistance in bacteria that rely strongly on β-lactamase expression
Alternative splicing: the pledge, the turn, and the prestige
No full text© The Author(s) 2017. This article is an open access publication. Open Access. This article is distributed under the terms of the Crea-tive Commons Attribution 4.0 International License (http://crea-tivecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.Alternative pre-mRNA splicing is a tightly controlled process conducted by the spliceosome, with the assistance of several regulators, resulting in the expression of different transcript isoforms from the same gene and increasing both transcriptome and proteome complexity. The differences between alternative isoforms may be subtle but enough to change the function or localization of the translated proteins. A fine control of the isoform balance is, therefore, needed throughout developmental stages and adult tissues or physiological conditions and it does not come as a surprise that several diseases are caused by its deregulation. In this review, we aim to bring the splicing machinery on stage and raise the curtain on its mechanisms and regulation throughout several systems and tissues of the human body, from neurodevelopment to the interactions with the human microbiome. We discuss, on one hand, the essential role of alternative splicing in assuring tissue function, diversity, and swiftness of response in these systems or tissues, and on the other hand, what goes wrong when its regulatory mechanisms fail. We also focus on the possibilities that splicing modulation therapies open for the future of personalized medicine, along with the leading techniques in this field. The final act of the spliceosome, however, is yet to be fully revealed, as more knowledge is needed regarding the complex regulatory network that coordinates alternative splicing and how its dysfunction leads to disease.The authors are supported by: EMBO Installation Grant (3057), Investigador FCT Starting Grant (IF/00595/2014) and iMM Lisboa start-up funds to NLB-M; Postdoctoral fellowships by UNESCO-L’Oreal For Women in Science Program (ERI/NCS/FLP/CDC.13.94) and iMM/FCT/MEC/FEDER (IMM/BPD/45-2016, LISBOA-01-0145-FEDER-007391) to LG-P; Fundação para a Ciência e Tecnologia (FCT) PhD fellowships to MCB (PD/BD/105854/2014) and MA-F (PD/BD/128283/2017); Fundação AstraZeneca Innovate Competition Award to MA-F.info:eu-repo/semantics/publishedVersio
EXPRESSION OF GLUTAMINE SYNTHETASE (GS) GENES IN DURUM WHEAT CULTIVARS CHARACTERIZED BY A DIFFERENT GRAIN PROTEIN CONTENT
No full textGlutamine synthetase (GS) is a key enzyme for nitrogen (N) assimilation in plants, which catalyses the ATP-dependent condensation of ammonium and glutamate into glutamine, the principal precursor for the synthesis of most nitrogenous cellular compounds. Glutamine synthetase exists in different isoforms classified into groups according to their localization within the cell: the cytosolic form, GS1, and the chloroplastic form, GS2. GS1 is responsible for assimilating the ammonium produced by reduction of nitrate in roots, and for synthesizing Gln for the transport of N between different organs, while the major function of GS2 is to reassimilate ammonia endogenously released by photorespiration. The goal of the present study was to assess a specific and reliable protocol of RT-real time PCR for the study of the GS genes differential expression in two durum wheat cultivars Ciccio and Svevo, characterized respectively by high and low kernel protein content. The expression study was conducted on several tissues and variable developmental stages. In particular, total RNA was extracted and cDNA synthesized from leaves and roots collected from ten wheat plants at different phonological stages, from branching to grain filling. In order to optimize the PCR reaction conditions, a set of six housekeeping genes represented by the selected sequences of Actin, α-tubulin, TEF-1α, ADP-RF, RLI and CDC, were assessed by preliminary qRT-PCR assays. Some discrepancies were observed in the ranking of the candidate reference genes, and three of them (RLI, CDC and ADP-RF), which appeared more effective, were chosen for the study of GS genes. Real Time PCR conducted for plastidic GS2-A2 and GS2-B2 genes, and for cytosolic GSe1 and GSe2 genes, showed a different expression pattern in the various developmental stages for both cvs. Ciccio and Svevo
Inhibitors of CLK protein kinases suppress cell growth and induce apoptosis by modulating pre-mRNA splicing.
No full textAccumulating evidence has demonstrated the importance of alternative splicing in various physiological processes, including the development of different diseases. CDC-like kinases (CLKs) and serine-arginine protein kinases (SRPKs) are components of the splicing machinery that are crucial for exon selection. The discovery of small molecule inhibitors against these kinases is of significant value, not only to delineate the molecular mechanisms of splicing, but also to identify potential therapeutic opportunities. Here we describe a series of small molecules that inhibit CLKs and SRPKs and thereby modulate pre-mRNA splicing. Treatment with these small molecules (Cpd-1, Cpd-2, or Cpd-3) significantly reduced the levels of endogenous phosphorylated SR proteins and caused enlargement of nuclear speckles in MDA-MB-468 cells. Additionally, the compounds resulted in splicing alterations of RPS6KB1 (S6K), and subsequent depletion of S6K protein. Interestingly, the activity of compounds selective for CLKs was well correlated with the activity for modulating S6K splicing as well as growth inhibition of cancer cells. A comprehensive mRNA sequencing approach revealed that the inhibitors induced splicing alterations and protein depletion for multiple genes, including those involved in growth and survival pathways such as S6K, EGFR, EIF3D, and PARP. Fluorescence pulse-chase labeling analyses demonstrated that isoforms with premature termination codons generated after treatment with the CLK inhibitors were degraded much faster than canonical mRNAs. Taken together, these results suggest that CLK inhibitors exhibit growth suppression and apoptosis induction through splicing alterations in genes involved in growth and survival. These small molecule inhibitors may be valuable tools for elucidating the molecular machinery of splicing and for the potential development of a novel class of antitumor agents
Characterisation of mice deficient for the pro-survival BCL-2 family member A1/BFL-1
No full text© 2018 Dr. Robyn Leigh SchenkApoptosis is important for the development and maintenance of a healthy immune system. The intrinsic apoptotic pathway is regulated by the BCL-2 family of proteins, which consists of both pro-survival and pro-apoptotic members. The balance between these two sub-groups determines whether a cell lives or dies. The levels of different BCL-2 family proteins is influenced by the signals that an immune cell receives – for example, cytokine signalling drives pro-survival protein expression, whilst cellular stress from cytokine deprivation upregulates pro-apoptotic proteins.
Studies using knockout mouse models have highlighted the importance of some of the pro-survival proteins in different haematopoietic cell types, such as BCL-2 in mature lymphocytes and MCL-1 in haematopoietic stem cells. The pro-survival protein A1/BFL-1 is highly expressed in many different immune cell subsets and is upregulated after immune cell activation. However, little is known about the physiological importance of A1/BFL-1 in the immune system. This is because the presence of three murine A1 isoforms complicates the generation of a knockout mouse model. We have generated a completely A1-deficient mouse strain through sequential gene targeting in embryonic stem cells. This thesis presents an analysis of A1’s role in the haematopoietic system through the study of these A1-/- mice.
A1/BFL-1 is reportedly expressed in B and T lymphocytes, neutrophils, mast cells, and conventional dendritic cells (cDCs). We characterised the cell numbers for these populations in the A1-/- mice in the steady state. There were no major differences found when compared to wild-type mice, although there was a small but significant decrease in cDC numbers in the spleen, and also small reductions in memory T cell populations. Given that A1/BFL-1 is upregulated by immune cell activation, we proceeded with in vitro activation assays with the various different immune cell subsets. Despite the reduction in memory T cells, in vitro survival of T cells after activation was unperturbed. A1-/- cDCs, however, had a marked survival disadvantage in tissue culture. We further characterised the A1-/- mice in response to immune challenge in vivo, with influenza infection, LCMV infection, and T cell-dependent immunization. The A1-/- mice responded normally to all of these models.
We reasoned that A1/BFL-1 may have overlapping roles with other pro-survival BCL-2 family proteins. To this end, we generated compound mutant mice that are deficient for A1 and heterozygous for BCL-2, BCL-X or MCL-1. We focused on the lymphocytic compartment of these mice, based on the co-expression of these pro-survival proteins in B and T lymphocytes. Again, we found no overt differences in the compound mutant mice when compared to control mice. It is likely that complete deletion of BCL-2/BCL-X/MCL-1 in combination with A1 loss is required to decipher these overlapping roles.
Overall, despite high expression of A1/BFL-1 in different immune cell types, loss of A1 is tolerable in mice and, therefore, A1 is a redundant pro-survival BCL-2 family member in the haematopoietic system
