ResearchPad - phosphatases https://www.researchpad.co Default RSS Feed en-us © 2020 Newgen KnowledgeWorks <![CDATA[Staurosporine and NEM mainly impair WNK-SPAK/OSR1 mediated phosphorylation of KCC2 and NKCC1]]> https://www.researchpad.co/article/elastic_article_14752 The pivotal role of KCC2 and NKCC1 in development and maintenance of fast inhibitory neurotransmission and their implication in severe human diseases arouse interest in posttranscriptional regulatory mechanisms such as (de)phosphorylation. Staurosporine (broad kinase inhibitor) and N-ethylmalemide (NEM) that modulate kinase and phosphatase activities enhance KCC2 and decrease NKCC1 activity. Here, we investigated the regulatory mechanism for this reciprocal regulation by mass spectrometry and immunoblot analyses using phospho-specific antibodies. Our analyses revealed that application of staurosporine or NEM dephosphorylates Thr1007 of KCC2, and Thr203, Thr207 and Thr212 of NKCC1. Dephosphorylation of Thr1007 of KCC2, and Thr207 and Thr212 of NKCC1 were previously demonstrated to activate KCC2 and to inactivate NKCC1. In addition, application of the two agents resulted in dephosphorylation of the T-loop and S-loop phosphorylation sites Thr233 and Ser373 of SPAK, a critical kinase in the WNK-SPAK/OSR1 signaling module mediating phosphorylation of KCC2 and NKCC1. Taken together, these results suggest that reciprocal regulation of KCC2 and NKCC1 via staurosporine and NEM is based on WNK-SPAK/OSR1 signaling. The key regulatory phospho-site Ser940 of KCC2 is not critically involved in the enhanced activation of KCC2 upon staurosporine and NEM treatment, as both agents have opposite effects on its phosphorylation status. Finally, NEM acts in a tissue-specific manner on Ser940, as shown by comparative analysis in HEK293 cells and immature cultured hippocampal neurons. In summary, our analyses identified phospho-sites that are responsive to staurosporine or NEM application. This provides important information towards a better understanding of the cooperative interactions of different phospho-sites.

]]>
<![CDATA[Prolyl isomerization of FAAP20 catalyzed by PIN1 regulates the Fanconi anemia pathway]]> https://www.researchpad.co/article/5c784fbdd5eed0c484007497

The Fanconi Anemia (FA) pathway is a multi-step DNA repair process at stalled replication forks in response to DNA interstrand cross-links (ICLs). Pathological mutation of key FA genes leads to the inherited disorder FA, characterized by progressive bone marrow failure and cancer predisposition. The study of FA is of great importance not only to children suffering from FA but also as a model to study cancer pathogenesis in light of genome instability among the general population. FANCD2 monoubiquitination by the FA core complex is an essential gateway that connects upstream DNA damage signaling to enzymatic steps of repair. FAAP20 is a key component of the FA core complex, and regulated proteolysis of FAAP20 mediated by the ubiquitin E3 ligase SCFFBW7 is critical for maintaining the integrity of the FA complex and FA pathway signaling. However, upstream regulatory mechanisms that govern this signaling remain unclear. Here, we show that PIN1, a phosphorylation-specific prolyl isomerase, regulates the integrity of the FA core complex, thus FA pathway activation. We demonstrate that PIN1 catalyzes cis-trans isomerization of the FAAP20 pSer48-Pro49 motif and promotes FAAP20 stability. Mechanistically, PIN1-induced conformational change of FAAP20 enhances its interaction with the PP2A phosphatase to counteract SCFFBW7-dependent proteolytic signaling at the phosphorylated degron motif. Accordingly, PIN1 deficiency impairs FANCD2 activation and the DNA ICL repair process. Together, our study establishes PIN1-dependent prolyl isomerization as a new regulator of the FA pathway and genomic integrity.

]]>
<![CDATA[Reprogramming of Trypanosoma cruzi metabolism triggered by parasite interaction with the host cell extracellular matrix]]> https://www.researchpad.co/article/5c648d19d5eed0c484c81fa6

Trypanosoma cruzi, the etiological agent of Chagas’ disease, affects 8 million people predominantly living in socioeconomic underdeveloped areas. T. cruzi trypomastigotes (Ty), the classical infective stage, interact with the extracellular matrix (ECM), an obligatory step before invasion of almost all mammalian cells in different tissues. Here we have characterized the proteome and phosphoproteome of T. cruzi trypomastigotes upon interaction with ECM (MTy) and the data are available via ProteomeXchange with identifier PXD010970. Proteins involved with metabolic processes (such as the glycolytic pathway), kinases, flagellum and microtubule related proteins, transport-associated proteins and RNA/DNA binding elements are highly represented in the pool of proteins modified by phosphorylation. Further, important metabolic switches triggered by this interaction with ECM were indicated by decreases in the phosphorylation of hexokinase, phosphofructokinase, fructose-2,6-bisphosphatase, phosphoglucomutase, phosphoglycerate kinase in MTy. Concomitantly, a decrease in the pyruvate and lactate and an increase of glucose and succinate contents were detected by GC-MS. These observations led us to focus on the changes in the glycolytic pathway upon binding of the parasite to the ECM. Inhibition of hexokinase, pyruvate kinase and lactate dehydrogenase activities in MTy were observed and this correlated with the phosphorylation levels of the respective enzymes. Putative kinases involved in protein phosphorylation altered upon parasite incubation with ECM were suggested by in silico analysis. Taken together, our results show that in addition to cytoskeletal changes and protease activation, a reprogramming of the trypomastigote metabolism is triggered by the interaction of the parasite with the ECM prior to cell invasion and differentiation into amastigotes, the multiplicative intracellular stage of T. cruzi in the vertebrate host.

]]>
<![CDATA[Dissection of the regulatory role for the N-terminal domain in Candida albicans protein phosphatase Z1]]> https://www.researchpad.co/article/5c5df31ad5eed0c484580d1d

The novel type, fungus specific protein phosphatase Z1 of the opportunistic pathogen, Candida albicans (CaPpz1) has several important physiological roles. It consists of a conserved C-terminal catalytic domain and a variable, intrinsically disordered, N-terminal regulatory domain. To test the function of these domains we modified the structure of CaPpz1 by in vitro mutagenesis. The two main domains were separated, four potential protein binding regions were deleted, and the myristoylation site as well as the active site of the enzyme was crippled by point mutations G2A and R262L, respectively. The in vitro phosphatase activity assay of the bacterially expressed recombinant proteins indicated that the N-terminal domain was inactive, while the C-terminal domain became highly active against myosin light chain substrate. The deletion of the N-terminal 1–16 amino acids and the G2A mutation significantly decreased the specific activity of the enzyme. Complementation of the ppz1 Saccharomyces cerevisiae deletion mutant strain with the different CaPpz1 forms demonstrated that the scission of the main domains, the two point mutations and the N-terminal 1–16 deletion rendered the phosphatase incompetent in the in vivo assays of LiCl tolerance and caffeine sensitivity. Thus our results confirmed the functional role of the N-terminal domain and highlighted the significance of the very N-terminal part of the protein in the regulation of CaPpz1.

]]>
<![CDATA[Surface molecules of extracellular vesicles secreted by the helminth pathogen Fasciola hepatica direct their internalisation by host cells]]> https://www.researchpad.co/article/5c4b7f2dd5eed0c484840b72

Helminth parasites secrete extracellular vesicles (EVs) that can be internalised by host immune cells resulting in modulation of host immunity. While the molecular cargo of EVs have been characterised in many parasites, little is known about the surface-exposed molecules that participate in ligand-receptor interactions with the host cell surface to initiate vesicle docking and subsequent internalisation. Using a membrane-impermeable biotin reagent to capture proteins displayed on the outer membrane surface of two EV sub-populations (termed 15k and 120k EVs) released by adult F. hepatica, we describe 380 surface proteins including an array of virulence factors, membrane transport proteins and molecules involved in EV biogenesis/trafficking. Proteomics and immunohistochemical analysis show that the 120k EVs have an endosomal origin and may be released from the parasite via the protonephridial (excretory) system whilst the larger 15k EVs are released from the gastrodermal epithelial cells that line the fluke gut. A parallel lectin microarray strategy was used to profile the topology of major surface oligosaccharides of intact fluorogenically-labelled EVs as they would be displayed to the host. Lectin profiles corresponding to glycoconjugates exposed on the surface of the 15 K and 120K EV sub-populations are practically identical but are distinct from those of the parasite surface tegument, although all are predominated by high mannose sugars. We found that while the F. hepatica EVs were resistant to exo- and endo-glycosidases, the glyco-amidase PNGase F drastically remodelled the surface oligosaccharides and blocked the uptake of EVs by host macrophages. In contrast, pre-treatment with antibodies obtained from infected hosts, or purified antibodies raised against the extracellular domains of specific EV surface proteins (DM9-containing protein, CD63 receptor and myoferlin), significantly enhanced their cellular internalisation. This work highlights the diversity of EV biogenesis and trafficking pathways used by F. hepatica and sheds light on the molecular interaction between parasite EVs and host cells.

]]>
<![CDATA[The Syk inhibitor R406 is a modulator of P-glycoprotein (ABCB1)-mediated multidrug resistance]]> https://www.researchpad.co/article/5c50c48ed5eed0c4845e88f0

In a previously published study, higher levels of spleen tyrosine kinase (Syk) were observed in recurrent post-chemotherapy ovarian cancers compared to primary tumors. Syk inhibition was found to stabilize microtubules and potentiate paclitaxel activity in cellular models of taxane-resistant ovarian cancers. We further studied the effects of Syk inhibition on paclitaxel activity in Syk(+) ovarian cancer cell models and in variants selected for taxane resistance. Syk inhibition was accomplished using RNAi and by exposure to the small molecule competitive inhibitor R406, the active metabolite of fostamatinib. Exposure to R406 or to a SYK-specific pool of siRNAs did not alter taxane activity in the OVCAR-3 cell line, which has the most Syk content in our panel of nine human ovarian cancer cell lines. However, treatment with R406 sensitised the multidrug resistant (MDR) variants MES-SA/Dx5 and SK-OV-3/TR to paclitaxel in a dose-dependent manner resulting from the inhibition of the ABCB1/P-glycoprotein (P-gp) drug transporter. These observations are Syk-independent since both MDR cell models are Syk negative. R406 modulated resistance to other known P-gp substrates, and we observed orthovanadate-sensitive ATPase stimulation resulting from treatment with R406. These data indicate that the chemo-sensitizing effect of R406 in taxane-resistant cells previously reported was not associated with Syk but resulted from the modulation of P-gp-mediated MDR.

]]>
<![CDATA[Hepatitis B virus core protein phosphorylation: Identification of the SRPK1 target sites and impact of their occupancy on RNA binding and capsid structure]]> https://www.researchpad.co/article/5c23f30fd5eed0c48404a273

Hepatitis B virus (HBV) replicates its 3 kb DNA genome through capsid-internal reverse transcription, initiated by assembly of 120 core protein (HBc) dimers around a complex of viral pregenomic (pg) RNA and polymerase. Following synthesis of relaxed circular (RC) DNA capsids can be enveloped and secreted as stable virions. Upon infection of a new cell, however, the capsid disintegrates to release the RC-DNA into the nucleus for conversion into covalently closed circular (ccc) DNA. HBc´s interactions with nucleic acids are mediated by an arginine-rich C terminal domain (CTD) with intrinsically strong non-specific RNA binding activity. Adaptation to the changing demands for nucleic acid binding during the viral life cycle is thought to involve dynamic phosphorylation / dephosphorylation events. However, neither the relevant enzymes nor their target sites in HBc are firmly established. Here we developed a bacterial coexpression system enabling access to definably phosphorylated HBc. Combining Phos-tag gel electrophoresis, mass spectrometry and mutagenesis we identified seven of the eight hydroxy amino acids in the CTD as target sites for serine-arginine rich protein kinase 1 (SRPK1); fewer sites were phosphorylated by PKA and PKC. Phosphorylation of all seven sites reduced nonspecific RNA encapsidation as drastically as deletion of the entire CTD and altered CTD surface accessibility, without major structure changes in the capsid shell. The bulk of capsids from human hepatoma cells was similarly highly, yet non-identically, phosphorylated as by SRPK1. While not proving SRPK1 as the infection-relevant HBc kinase the data suggest a mechanism whereby high-level HBc phosphorylation principally suppresses RNA binding whereas one or few strategic dephosphorylation events enable selective packaging of the pgRNA/polymerase complex. The tools developed in this study should greatly facilitate the further deciphering of the role of HBc phosphorylation in HBV infection and its evaluation as a potential new therapeutic target.

]]>
<![CDATA[Physiological relevance of epithelial geometry: New insights into the standing gradient model and the role of LI cadherin]]> https://www.researchpad.co/article/5c26976dd5eed0c48470f804

We introduce a mathematical model of an absorbing leaky epithelium to reconsider the problem formulated by Diamond and Bossert in 1967: whether “… some distinctive physiological properties of epithelia might arise as geometrical consequences of epithelial ultrastructure”. A standing gradient model of the intercellular cleft (IC) is presented that includes tight junctions (TJ) and ion channels uniformly distributed along the whole cleft. This nonlinear system has an intrinsic homogeneous concentration and the spatial scale necessary to establish it along the cleft. These parameters have not been elucidated so far. We further provide non-perturbative analytical approximations for a broad range of parameters. We found that narrowing of the IC increases ion concentration dramatically and can therefore prevent outflow through tight junctions (TJs) and the lateral membrane, as long as extremely high luminal osmolarities are not reached. Our model predicts that the system is to some extent self-regulating and thereby prevents fluxes into the lumen. Recent experimental evidence has shown that liver-intestine (LI) cadherin can control the up/down flux in intestines via regulation of the cleft width. This finding is in full agreement with predictions of our model. We suggest that LI-cadherin may increase water transport through epithelia via sequential narrowing of the cleft, starting from the highest concentration area at the beginning of the cleft and triggering a propagating squeezing motion.

]]>
<![CDATA[Mek1 coordinates meiotic progression with DNA break repair by directly phosphorylating and inhibiting the yeast pachytene exit regulator Ndt80]]> https://www.researchpad.co/article/5c099408d5eed0c4842ae123

Meiotic recombination plays a critical role in sexual reproduction by creating crossovers between homologous chromosomes. These crossovers, along with sister chromatid cohesion, connect homologs to enable proper segregation at Meiosis I. Recombination is initiated by programmed double strand breaks (DSBs) at particular regions of the genome. The meiotic recombination checkpoint uses meiosis-specific modifications to the DSB-induced DNA damage response to provide time to convert these breaks into interhomolog crossovers by delaying entry into Meiosis I until the DSBs have been repaired. The meiosis-specific kinase, Mek1, is a key regulator of meiotic recombination pathway choice, as well as being required for the meiotic recombination checkpoint. The major target of this checkpoint is the meiosis-specific transcription factor, Ndt80, which is essential to express genes necessary for completion of recombination and meiotic progression. The molecular mechanism by which cells monitor meiotic DSB repair to allow entry into Meiosis I with unbroken chromosomes was unknown. Using genetic and biochemical approaches, this work demonstrates that in the presence of DSBs, activated Mek1 binds to Ndt80 and phosphorylates the transcription factor, thus inhibiting DNA binding and preventing Ndt80’s function as a transcriptional activator. Repair of DSBs by recombination reduces Mek1 activity, resulting in removal of the inhibitory Mek1 phosphates. Phosphorylation of Ndt80 by the meiosis-specific kinase, Ime2, then results in fully activated Ndt80. Ndt80 upregulates transcription of its own gene, as well as target genes, resulting in prophase exit and progression through meiosis.

]]>
<![CDATA[Treatment with XAV-939 prevents in vitro calcification of human valvular interstitial cells]]> https://www.researchpad.co/article/5c141f05d5eed0c484d294ae

The development of a substance or inhibitor-based treatment strategy for the prevention of aortic valve stenosis is a challenge and a main focus of medical research in this area. One strategy may be to use the tankyrase inhibitor XAV-939, which leads to Axin stabilisation and subsequent destruction of the β-catenin complex and dephosphorylation of β-catenin. The dephosphorylated active form of β-catenin (non-phospho-β-catenin) then promotes nuclear transcription that leads to osteogenesis. The aims of the present study were to develop an experimental system for inducing in vitro calcification of human aortic valvular interstitial cells (VICs) to investigate the potential anti-calcific effect of XAV-939 and to analyse expression of the Wnt signalling proteins and Sox9, a chondrogenesis regulator, in this model. Calcification of human VIC cultures was induced by cultivation in an osteogenic medium and the effect of co-incubation with 1μM XAV-939 was monitored. Calcification was quantified when mineral deposits were visible in culture and was histologically verified by von Kossa or Alizarin red staining and by IR-spectroscopy. Protein expression of alkaline phosphatase, Axin, β-catenin and Sox9 were quantified by western blotting. In 58% of the VIC preparations, calcification was induced in an osteogenic culture medium and was accompanied by upregulation of alkaline phosphatase. The calcification induction was prevented by the XAV-939 co-treatment and the alkaline phosphatase upregulation was suppressed. As expected, Axin was upregulated, but the levels of active non-phospho-β-catenin were also enhanced. Sox9 was induced during XAV-939 treatment but apparently not as a result of downregulation of β-catenin signalling. XAV-939 was therefore able to prevent calcification of human VIC cultures, and XAV-939 treatment was accompanied by upregulation of active non-phospho-β-catenin. Although XAV-939 does not downregulate active β-catenin, treatment with XAV-939 results in Sox9 upregulation that may prevent the calcification process.

]]>
<![CDATA[Comparative transcriptome profiling of Blumeria graminis f. sp. tritici during compatible and incompatible interactions with sister wheat lines carrying and lacking Pm40]]> https://www.researchpad.co/article/5b4a196b463d7e428027f8b2

Blumeria graminis f. sp. tritici (Bgt) is an obligate biotrophic fungus that causes wheat powdery mildew, which is a devastating disease in wheat. However, little is known about the pathogenesis of this fungus, and differences in the pathogenesis of the same pathogen at various resistance levels in hosts have not been determined. In the present study, leaf tissues of both Pm40-expressing hexaploid wheat line L658 and its Pm40-deficient sister line L958 were harvested at 0 (without inoculation), 6, 12, 24, 48 and 72 hours post-inoculation (hpi) with Bgt race 15 and then subjected to RNA sequencing (RNA-seq). In addition, we also observed changes in fungal growth morphology at the aforementioned time points. There was a high correlation between percentage of reads mapped to the Bgt reference genome and biomass of the fungus within the leaf tissue during the growth process. The percentage of mapped reads of Bgt in compatible interactions was significantly higher (at the p<0.05 level) than that of reads in incompatible interactions from 24 to 72 hpi. Further functional annotations indicated that expression levels of genes encoding H+-transporting ATPase, putative secreted effector proteins (PSEPs) and heat shock proteins (HSPs) were significantly up-regulated in compatible interactions compared with these levels in incompatible interactions, particularly at 72 hpi. Moreover, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis suggested that genes involved in the endocytosis pathway were also enriched in compatible interactions. Overall, genes encoding H+-transporting ATPase, PSEPs and HSPs possibly played crucial roles in successfully establishing the pathogenesis of compatible interactions during late stages of inoculation. The study results also indicated that endocytosis is likely to play a potential role in Bgt in establishing compatible interactions.

]]>
<![CDATA[Enzyme sequestration by the substrate: An analysis in the deterministic and stochastic domains]]> https://www.researchpad.co/article/5b07d0f1463d7e0d4a37a6f4

This paper is concerned with the potential multistability of protein concentrations in the cell. That is, situations where one, or a family of, proteins may sit at one of two or more different steady state concentrations in otherwise identical cells, and in spite of them being in the same environment. For models of multisite protein phosphorylation for example, in the presence of excess substrate, it has been shown that the achievable number of stable steady states can increase linearly with the number of phosphosites available. In this paper, we analyse the consequences of adding enzyme docking to these and similar models, with the resultant sequestration of phosphatase and kinase by the fully unphosphorylated and by the fully phosphorylated substrates respectively. In the large molecule numbers limit, where deterministic analysis is applicable, we prove that there are always values for these rates of sequestration which, when exceeded, limit the extent of multistability. For the models considered here, these numbers are much smaller than the affinity of the enzymes to the substrate when it is in a modifiable state. As substrate enzyme-sequestration is increased, we further prove that the number of steady states will inevitably be reduced to one. For smaller molecule numbers a stochastic analysis is more appropriate, where multistability in the large molecule numbers limit can manifest itself as multimodality of the probability distribution; the system spending periods of time in the vicinity of one mode before jumping to another. Here, we find that substrate enzyme sequestration can induce bimodality even in systems where only a single steady state can exist at large numbers. To facilitate this analysis, we develop a weakly chained diagonally dominant M-matrix formulation of the Chemical Master Equation, allowing greater insights in the way particular mechanisms, like enzyme sequestration, can shape probability distributions and therefore exhibit different behaviour across different regimes.

]]>
<![CDATA[A secreted Heat shock protein 90 of Trichomonas vaginalis]]> https://www.researchpad.co/article/5b079d49463d7e75962e790c

Trichomonas vaginalis is a causative agent of Trichomoniasis, a leading non-viral sexually transmitted disease worldwide. In the current study, we show Heat shock protein 90 is essential for its growth. Upon genomic analysis of the parasite, it was found to possess seven ORFs which could potentially encode Hsp90 isoforms. We identified a cytosolic Hsp90 homolog, four homologs which can align to truncated cytosolic Hsp90 gene products along with two Grp94 homologs (ER isoform of Hsp90). However, both Grp94 orthologs lacked an ER retention motif. In cancer cells, it is very well established that Hsp90 is secreted and regulates key clients involved in metastases, migration, and invasion. Since Trichomonas Grp94 lacks ER retention motif, we examined the possibility of its secretion. By using cell biology and biochemical approaches we show that the Grp94 isoform of Hsp90 is secreted by the parasite by the classical ER-Golgi pathway. This is the first report of a genome encoded secreted Hsp90 in a clinically important parasitic protozoan.

]]>
<![CDATA[The SecA2 pathway of Mycobacterium tuberculosis exports effectors that work in concert to arrest phagosome and autophagosome maturation]]> https://www.researchpad.co/article/5af106c6463d7e336df9e53f

To subvert host defenses, Mycobacterium tuberculosis (Mtb) avoids being delivered to degradative phagolysosomes in macrophages by arresting the normal host process of phagosome maturation. Phagosome maturation arrest by Mtb involves multiple effectors and much remains unknown about this important aspect of Mtb pathogenesis. The SecA2 dependent protein export system is required for phagosome maturation arrest and consequently growth of Mtb in macrophages. To better understand the role of the SecA2 pathway in phagosome maturation arrest, we identified two effectors exported by SecA2 that contribute to this process: the phosphatase SapM and the kinase PknG. Then, utilizing the secA2 mutant of Mtb as a platform to study effector functions, we identified specific steps in phagosome maturation inhibited by SapM and/or PknG. By identifying a histidine residue that is essential for SapM phosphatase activity, we confirmed for the first time that the phosphatase activity of SapM is required for its effects on phagosome maturation in macrophages. We further demonstrated that SecA2 export of SapM and PknG contributes to the ability of Mtb to replicate in macrophages. Finally, we extended our understanding of the SecA2 pathway, SapM, and PknG by revealing that their contribution goes beyond preventing Mtb delivery to mature phagolysosomes and includes inhibiting Mtb delivery to autophagolysosomes. Together, our results revealed SapM and PknG to be two effectors exported by the SecA2 pathway of Mtb with distinct as well as cumulative effects on phagosome and autophagosome maturation. Our results further reveal that Mtb must have additional mechanisms of limiting acidification of the phagosome, beyond inhibiting recruitment of the V-ATPase proton pump to the phagosome, and they indicate differences between effects of Mtb on phagosome and autophagosome maturation.

]]>
<![CDATA[Fine-Tuning of Pten Localization and Phosphatase Activity Is Essential for Zebrafish Angiogenesis]]> https://www.researchpad.co/article/5989da30ab0ee8fa60b840a4

The lipid- and protein phosphatase PTEN is an essential tumor suppressor that is highly conserved among all higher eukaryotes. As an antagonist of the PI3K/Akt cell survival and proliferation pathway, it exerts its most prominent function at the cell membrane, but (PIP3-independent) functions of nuclear PTEN have been discovered as well. PTEN subcellular localization is tightly controlled by its protein conformation. In the closed conformation, PTEN localizes predominantly to the cytoplasm. Opening up of the conformation of PTEN exposes N-terminal and C-terminal regions of the protein that are required for both interaction with the cell membrane and translocation to the nucleus. Lack of Pten leads to hyperbranching of the intersegmental vessels during zebrafish embryogenesis, which is rescued by expression of exogenous Pten. Here, we observed that expression of mutant PTEN with an open conformation rescued the hyperbranching phenotype in pten double homozygous embryos and suppressed the increased p-Akt levels that are characteristic for embryos lacking Pten. In addition, in pten mutant and wild type embryos alike, open conformation PTEN induced stalled intersegmental vessels, which fail to connect with the dorsal longitudinal anastomotic vessel. Functional hyperactivity of open conformation PTEN in comparison to wild type PTEN seems to result predominantly from its enhanced recruitment to the cell membrane. Enhanced recruitment of phosphatase inactive mutants to the membrane did not induce the stalled vessel phenotype nor did it rescue the hyperbranching phenotype in pten double homozygous embryos, indicating that PTEN phosphatase activity is indispensable for its regulatory function during angiogenesis. Taken together, our data suggest that PTEN phosphatase activity needs to be carefully fine-tuned for normal embryogenesis and that the control of its subcellular localization is a key mechanism in this process.

]]>
<![CDATA[Unidirectional Flux Balance of Monovalent Ions in Cells with Na/Na and Li/Na Exchange: Experimental and Computational Studies on Lymphoid U937 Cells]]> https://www.researchpad.co/article/5989db00ab0ee8fa60bc671f

Monovalent ion traffic across the cell membrane occurs via various pathways. Evaluation of individual fluxes in whole cell is hampered by their strong interdependence. This difficulty can be overcome by computational analysis of the whole cell flux balance. However, the previous computational studies disregarded ion movement of the self-exchange type. We have taken this exchange into account. The developed software allows determination of unidirectional fluxes of all monovalent ions via the major pathways both under the balanced state and during transient processes. We show how the problem of finding the rate coefficients can be solved by measurement of monovalent ion concentrations and some of the fluxes. Interdependence of fluxes due to the mandatory conditions of electroneutrality and osmotic balance and due to specific effects can be discriminated, enabling one to identify specific changes in ion transfer machinery under varied conditions. To test the effectiveness of the developed approach we made use of the fact that Li/Na exchange is known to be an analogue of the coupled Na/Na exchange. Thus, we compared the predicted and experimental data obtained on U937 cells under varied Li+ concentrations and following inhibition of the sodium pump with ouabain. We found that the coupled Na/Na exchange in U937 cells comprises a significant portion of the entire Na+ turnover. The data showed that the loading of the sodium pump by Li/Na exchange involved in the secondary active Li+ transport at 1–10 mM external Li+ is small. This result may be extrapolated to similar Li+ and Na+ flux relationships in erythrocytes and other cells in patients treated with Li+ in therapeutic doses. The developed computational approach is applicable for studying various cells and can be useful in education for demonstrating the effects of individual transporters and channels on ion gradients, cell water content and membrane potential.

]]>
<![CDATA[Dynamics robustness of cascading systems]]> https://www.researchpad.co/article/5989db54ab0ee8fa60bdd007

A most important property of biochemical systems is robustness. Static robustness, e.g., homeostasis, is the insensitivity of a state against perturbations, whereas dynamics robustness, e.g., homeorhesis, is the insensitivity of a dynamic process. In contrast to the extensively studied static robustness, dynamics robustness, i.e., how a system creates an invariant temporal profile against perturbations, is little explored despite transient dynamics being crucial for cellular fates and are reported to be robust experimentally. For example, the duration of a stimulus elicits different phenotypic responses, and signaling networks process and encode temporal information. Hence, robustness in time courses will be necessary for functional biochemical networks. Based on dynamical systems theory, we uncovered a general mechanism to achieve dynamics robustness. Using a three-stage linear signaling cascade as an example, we found that the temporal profiles and response duration post-stimulus is robust to perturbations against certain parameters. Then analyzing the linearized model, we elucidated the criteria of when signaling cascades will display dynamics robustness. We found that changes in the upstream modules are masked in the cascade, and that the response duration is mainly controlled by the rate-limiting module and organization of the cascade’s kinetics. Specifically, we found two necessary conditions for dynamics robustness in signaling cascades: 1) Constraint on the rate-limiting process: The phosphatase activity in the perturbed module is not the slowest. 2) Constraints on the initial conditions: The kinase activity needs to be fast enough such that each module is saturated even with fast phosphatase activity and upstream changes are attenuated. We discussed the relevance of such robustness to several biological examples and the validity of the above conditions therein. Given the applicability of dynamics robustness to a variety of systems, it will provide a general basis for how biological systems function dynamically.

]]>
<![CDATA[Minimal oscillating subnetwork in the Huang-Ferrell model of the MAPK cascade]]> https://www.researchpad.co/article/5989db5fab0ee8fa60be1166

Prompted by the recent growing evidence of oscillatory behavior involving MAPK cascades we present a systematic approach of analyzing models and elucidating the nature of biochemical oscillations based on reaction network theory. In particular, we formulate a minimal biochemically consistent mass action subnetwork of the Huang-Ferrell model of the MAPK signalling that provides an oscillatory response when a parameter controlling the activation of the top-tier kinase is varied. Such dynamics are either intertwined with or separated from the earlier found bistable/hysteretic behavior in this model. Using the theory of stability of stoichiometric networks, we reduce the original MAPK model, convert kinetic to convex parameters and examine those properties of the minimal subnetwork that underlie the oscillatory dynamics. We also use the methods of classification of chemical oscillatory networks to explain the rhythmic behavior in physicochemical terms, i.e., we identify of the role of individual biochemical species in positive and negative feedback loops and describe their coordinated action leading to oscillations. Our approach provides an insight into dynamics without the necessity of knowing rate coefficients and thus is useful prior the statistical evaluation of parameters.

]]>
<![CDATA[Drosophila DNA polymerase theta utilizes both helicase-like and polymerase domains during microhomology-mediated end joining and interstrand crosslink repair]]> https://www.researchpad.co/article/5989db5cab0ee8fa60be03c5

Double strand breaks (DSBs) and interstrand crosslinks (ICLs) are toxic DNA lesions that can be repaired through multiple pathways, some of which involve shared proteins. One of these proteins, DNA Polymerase θ (Pol θ), coordinates a mutagenic DSB repair pathway named microhomology-mediated end joining (MMEJ) and is also a critical component for bypass or repair of ICLs in several organisms. Pol θ contains both polymerase and helicase-like domains that are tethered by an unstructured central region. While the role of the polymerase domain in promoting MMEJ has been studied extensively both in vitro and in vivo, a function for the helicase-like domain, which possesses DNA-dependent ATPase activity, remains unclear. Here, we utilize genetic and biochemical analyses to examine the roles of the helicase-like and polymerase domains of Drosophila Pol θ. We demonstrate an absolute requirement for both polymerase and ATPase activities during ICL repair in vivo. However, similar to mammalian systems, polymerase activity, but not ATPase activity, is required for ionizing radiation-induced DSB repair. Using a site-specific break repair assay, we show that overall end-joining efficiency is not affected in ATPase-dead mutants, but there is a significant decrease in templated insertion events. In vitro, Pol θ can efficiently bypass a model unhooked nitrogen mustard crosslink and promote DNA synthesis following microhomology annealing, although ATPase activity is not required for these functions. Together, our data illustrate the functional importance of the helicase-like domain of Pol θ and suggest that its tethering to the polymerase domain is important for its multiple functions in DNA repair and damage tolerance.

]]>
<![CDATA[Isolation Driven Divergence in Osmoregulation in Galaxias maculatus (Jenyns, 1848) (Actinopterygii: Osmeriformes)]]> https://www.researchpad.co/article/5989d9d5ab0ee8fa60b65bc6

Background

Marine species have colonized extreme environments during evolution such as freshwater habitats. The amphidromous teleost fish, Galaxias maculatus is found mainly migrating between estuaries and rivers, but some landlocked populations have been described in lakes formed during the last deglaciation process in the Andes. In the present study we use mtDNA sequences to reconstruct the historical scenario of colonization of such a lake and evaluated the osmoregulatory shift associated to changes in habitat and life cycle between amphidromous and landlocked populations.

Results

Standard diversity indices including the average number of nucleotide differences (Π) and the haplotype diversity index (H) indicated that both populations were, as expected, genetically distinctive, being the landlocked population less diverse than the diadromous one. Similarly, pairwise GST and NST comparison detected statistically significant differences between both populations, while genealogy of haplotypes evidenced a recent founder effect from the diadromous stock, followed by an expansion process in the lake. To test for physiological differences, individuals of both populations were challenged with a range of salinities from 0 to 30 ppt for 8 days following a period of progressive acclimation. The results showed that the landlocked population had a surprisingly wider tolerance to salinity, as landlocked fish survival was 100% from 0 to 20 ppt, whereas diadromous fish survival was 100% only from 10 to 15 ppt. The activity of ATPase enzymes, including Na+/K+-ATPase (NKA), and H+-ATPase (HA) was measured in gills and intestine. Activity differences were detected between the populations at the lowest salinities, including differences in ATPases other than NKA and HA. Population differences in mortality are not reflected in enzyme activity differences, suggesting divergence in other processes.

Conclusions

These results clearly demonstrate the striking adaptive changes of G. maculatus osmoregulatory system, especially at hyposmotic environments, associated to a drastic shift in habitat and life cycle at a scale of a few thousand years.

]]>