Phosphorylated Peptide Derived from the Myosin Phosphatase Target Subunit Is a Novel Inhibitor of Protein Phosphatase-1.

Identification of specific protein phosphatase-1 (PP1) inhibitors is of special importance regarding the study of its cellular functions and may have therapeutic values in diseases coupled to signaling processes. In this study, we prove that a phosphorylated peptide of the inhibitory region of myosin phosphatase (MP) target subunit (MYPT1), R690QSRRS(pT696)QGVTL701 (P-Thr696-MYPT1690-701), interacts with and inhibits the PP1 catalytic subunit (PP1c, IC50 = 3.84 µM) and the MP holoenzyme (Flag-MYPT1-PP1c, IC50 = 3.84 µM). Saturation transfer difference NMR measurements established binding of hydrophobic and basic regions of P-Thr696-MYPT1690-701 to PP1c, suggesting interactions with the hydrophobic and acidic substrate binding grooves. P-Thr696-MYPT1690-701 was dephosphorylated by PP1c slowly (t1/2 = 81.6-87.9 min), which was further impeded (t1/2 = 103 min) in the presence of the phosphorylated 20 kDa myosin light chain (P-MLC20). In contrast, P-Thr696-MYPT1690-701 (10-500 µM) slowed down the dephosphorylation of P-MLC20 (t1/2 = 1.69 min) significantly (t1/2 = 2.49-10.06 min). These data are compatible with an unfair competition mechanism between the inhibitory phosphopeptide and the phosphosubstrate. Docking simulations of the PP1c-P-MYPT1690-701 complexes with phosphothreonine (PP1c-P-Thr696-MYPT1690-701) or phosphoserine (PP1c-P-Ser696-MYPT1690-701) suggested their distinct poses on the surface of PP1c. In addition, the arrangements and distances of the surrounding coordinating residues of PP1c around the phosphothreonine or phosphoserine at the active site were distinct, which may account for their different hydrolysis rate. It is presumed that P-Thr696-MYPT1690-701 binds tightly at the active center but the phosphoester hydrolysis is less preferable compared to P-Ser696-MYPT1690-701 or phosphoserine substrates. Moreover, the inhibitory phosphopeptide may serve as a template to synthesize cell permeable PP1-specific peptide inhibitors.

A Fucosylated Lactose-Presenting Tetravalent Glycocluster Acting as a Mutual Ligand of Pseudomonas aeruginosa Lectins A (PA-IL) and B (PA-IIL)-Synthesis and Interaction Studies.

The Gram-negative bacterium Pseudomonas aeruginosa is an important opportunistic human pathogen associated with cystic fibrosis. P. aeruginosa produces two soluble lectins, the d-galactose-specific lectin PA-IL (LecA) and the l-fucose-specific lectin PA-IIL (LecB), among other virulence factors. These lectins play an important role in the adhesion to host cells and biofilm formation. Moreover, PA-IL is cytotoxic to respiratory cells in the primary culture. Therefore, these lectins are promising therapeutic targets. Specifically, carbohydrate-based compounds could inhibit their activity. In the present work, a 3-O-fucosyl lactose-containing tetravalent glycocluster was synthesized and utilized as a mutual ligand of galactophilic and fucophilic lectins. Pentaerythritol equipped with azido ethylene glycol-linkers was chosen as a multivalent scaffold and the glycocluster was constructed by coupling the scaffold with propargyl 3-O-fucosyl lactoside using an azide-alkyne 1,3-dipolar cycloaddition reaction. The interactions between the glycocluster and PA-IL or PA-IIL were investigated by isothermal titration microcalorimetry and saturation transfer difference NMR spectroscopy. These results may assist in the development of efficient anti-adhesion therapy for the treatment of a P. aeruginosa infection.

Myosin Phosphatase Is Implicated in the Control of THP-1 Monocyte to Macrophage Differentiation.

Monocyte to macrophage differentiation is characterized by the activation of various signal transduction pathways, which may be modulated by protein phosphorylation; however, the impact of protein kinases and phosphatases is not well understood yet. It has been demonstrated that actomyosin rearrangement during macrophage differentiation is dependent on Rho-associated protein kinase (ROCK). Myosin phosphatase (MP) target subunit-1 (MYPT1) is one of the major cellular substrates of ROCK, and MP is often a counter enzyme of ROCK; therefore, MP may also control macrophage differentiation. Changes in MP activity and the effects of MP activation were studied on PMA or l,25(OH)2D3-induced differentiation of monocytic THP-1 cells. During macrophage differentiation, phosphorylation of MYPT1 at Thr696 and Thr853 increased significantly, resulting in inhibition of MP. The ROCK inhibitor H1152 and the MP activator epigallocatechin-3-gallate (EGCG) attenuated MYPT1 phosphorylation and concomitantly decreased the extent of phosphorylation of 20 kDa myosin light chain. H1152 and EGCG pretreatment also suppressed the expression of CD11b and weakened the PMA-induced adherence of the cells. Our results indicate that MP activation/inhibition contributes to the efficacy of monocyte to macrophage differentiation, and this enzyme may be a target for pharmacological interventions in the control of disease states that are affected by excessive macrophage differentiation.

Smoothelin-Like Protein 1 Regulates the Thyroid Hormone-Induced Homeostasis and Remodeling of C2C12 Cells via the Modulation of Myosin Phosphatase.

The pathological elevation of the active thyroid hormone (T3) level results in the manifestation of hyperthyroidism, which is associated with alterations in the differentiation and contractile function of skeletal muscle (SKM). Myosin phosphatase (MP) is a major cellular regulator that hydrolyzes the phosphoserine of phosphorylated myosin II light chain. MP consists of an MYPT1/2 regulatory and a protein phosphatase 1 catalytic subunit. Smoothelin-like protein 1 (SMTNL1) is known to inhibit MP by directly binding to MP as well as by suppressing the expression of MYPT1 at the transcriptional level. Supraphysiological vs. physiological concentration of T3 were applied on C2C12 myoblasts and differentiated myotubes in combination with the overexpression of SMTNL1 to assess the role and regulation of MP under these conditions. In non-differentiated myoblasts, MP included MYPT1 in the holoenzyme complex and its expression and activity was regulated by SMTNL1, affecting the phosphorylation level of MLC20 assessed using semi-quantitative Western blot analysis. SMTNL1 negatively influenced the migration and cytoskeletal remodeling of myoblasts measured by high content screening. In contrast, in myotubes, the expression of MYPT2 but not MYPT1 increased in a T3-dependent and SMTNL1-independent manner. T3 treatment combined with SMTNL1 overexpression impeded the activity of MP. In addition, MP interacted with Na+/K+-ATPase and dephosphorylated its inhibitory phosphorylation sites, identifying this protein as a novel MP substrate. These findings may help us gain a better understanding of myopathy, muscle weakness and the disorder of muscle regeneration in hyperthyroid patients.

Differential mechanisms of adenosine- and ATPγS-induced microvascular endothelial barrier strengthening.

Maintenance of the endothelial cell (EC) barrier is critical to vascular homeostasis and a loss of barrier integrity results in increased vascular permeability. While the mechanisms that govern increased EC permeability have been under intense investigation over the past several decades, the processes regulating the preservation/restoration of the EC barrier remain poorly understood. Herein we show that the extracellular purines, adenosine (Ado) and adenosine 5'-[γ-thio]-triphosphate (ATPγS) can strengthen the barrier function of human lung microvascular EC (HLMVEC). This ability involves protein kinase A (PKA) activation and decreases in myosin light chain 20 (MLC20) phosphorylation secondary to the involvement of MLC phosphatase (MLCP). In contrast to Ado, ATPγS-induced PKA activation is accompanied by a modest, but significant decrease in cyclic adenosine monophosphate (cAMP) levels supporting the existence of an unconventional cAMP-independent pathway of PKA activation. Furthermore, ATPγS-induced EC barrier strengthening does not involve the Rap guanine nucleotide exchange factor 3 (EPAC1) which is directly activated by cAMP but is instead dependent upon PKA-anchor protein 2 (AKAP2) expression. We also found that AKAP2 can directly interact with the myosin phosphatase-targeting protein MYPT1 and that depletion of AKAP2 abolished ATPγS-induced increases in transendothelial electrical resistance. Ado-induced strengthening of the HLMVEC barrier required the coordinated activation of PKA and EPAC1 in a cAMP-dependent manner. In summary, ATPγS-induced enhancement of the EC barrier is EPAC1-independent and is instead mediated by activation of PKA which is then guided by AKAP2, in a cAMP-independent mechanism, to activate MLCP which dephosphorylates MLC20 resulting in reduced EC contraction and preservation.

Inhibition of protein phosphatase-1 and -2A by ellagitannins: structure-inhibitory potency relationships and influences on cellular systems.

Several ellagitannins inhibited the activity of protein phosphatase-1 (PP1) and -2 A (PP2A) catalytic subunits (PP1c and PP2Ac) with preferential suppression of PP1c over PP2Ac. The inhibitory potency for PP1c followed the order of tellimagrandin I > mahtabin A > praecoxin B > 1.2-Di-O-galloyl-4.6-(S)-HHDP-ß-D-glucopyranose > pedunculagin with IC50 values ranging from 0.20 µM to 2.47 µM. The interaction of PP1c and tellimagrandin I was assessed by NMR saturation transfer difference, surface plasmon resonance, isothermal titration calorimetry, and microscale thermophoresis based binding techniques. Tellimagrandin I suppressed viability and phosphatase activity of HeLa cells, while mahtabin A was without effect. Conversely, mahtabin A increased the phosphorylation level of SNAP-25Thr138 and suppressed exocytosis of cortical synaptosomes, whereas tellimagrandin I was without influence. Our results establish ellagitannins as partially selective inhibitors of PP1 and indicate that these polyphenols may act distinctly in cellular systems depending on their membrane permeability and/or their actions on cell membranes.

Tropomyosins Regulate the Severing Activity of Gelsolin in Isoform-Dependent and Independent Manners.

The actin cytoskeleton fulfills numerous key cellular functions, which are tightly regulated in activity, localization, and temporal patterning by actin binding proteins. Tropomyosins and gelsolin are two such filament-regulating proteins. Here, we investigate how the effects of tropomyosins are coupled to the binding and activity of gelsolin. We show that the three investigated tropomyosin isoforms (Tpm1.1, Tpm1.12, and Tpm3.1) bind to gelsolin with micromolar or submicromolar affinities. Tropomyosin binding enhances the activity of gelsolin in actin polymerization and depolymerization assays. However, the effects of the three tropomyosin isoforms varied. The tropomyosin isoforms studied also differed in their ability to protect pre-existing actin filaments from severing by gelsolin. Based on the observed specificity of the interactions between tropomyosins, actin filaments, and gelsolin, we propose that tropomyosin isoforms specify which populations of actin filaments should be targeted by, or protected from, gelsolin-mediated depolymerization in living cells.

Aralkyl selenoglycosides and related selenosugars in acetylated form activate protein phosphatase-1 and -2A.

Aralkyl and aryl selenoglycosides as well as glycosyl selenocarboxylate derivatives were assayed on the activity of protein phosphatase-1 (PP1) and -2A (PP2A) catalytic subunits (PP1c and PP2Ac) in search of compounds for PP1c and PP2Ac effectors. The majority of tested selenoglycosides activated both PP1c and PP2Ac by ∼2-4-fold in a phosphatase assay with phosphorylated myosin light chain substrate when the hydroxyl groups of the glycosyl moiety were acetylated, but they were without any effects in the non-acetylated forms. A peptide from the myosin phosphatase target subunit-1 (MYPT123-38) that included an RVxF PP1c-binding motif attenuated activation of PP1c by 2-Trifluoromethylbenzyl 2,3,4,6-tetra-O-acetyl-1-seleno-ß-d-glucopyranoside (TFM-BASG) and 4-Bromobenzyl 2,3,4,6-tetra-O-acetyl-1-seleno-ß-d-glucopyranoside (Br-BASG). MYPT123-38 stimulated PP2Ac and contributed to PP2Ac activation exerted by either Br-BASG or TFM-BASG. Br-BASG and TFM-BASG suppressed partially binding of PP1c to MYPT1 in surface plasmon resonance based binding experiments. Molecular docking predicted that the hydrophobic binding surfaces in PP1c for interaction with either the RVxF residues of PP1c-interactors or selenoglycosides are partially overlapped. Br-BASG and TFM-BASG caused a moderate increase in the phosphatase activity of HeLa cells in 1 h, and suppressed cell viability in 24 h incubations. In conclusion, our present study identified selenoglycosides as novel activators of PP1 and PP2A as well as provided insights into the structural background of their interactions establishing a molecular model for future design of more efficient phosphatase activator molecules.

Protein phosphatase-1 is involved in the maintenance of normal homeostasis and in UVA irradiation-induced pathological alterations in HaCaT cells and in mouse skin.

The number of ultraviolet (UV) radiation-induced skin diseases such as melanomas is on the rise. The altered behavior of keratinocytes is often coupled with signaling events in which Ser/Thr specific protein kinases and phosphatases regulate various cellular functions. In the present study the role of protein phosphatase-1 (PP1) was investigated in the response of human keratinocyte (HaCaT) cells and mouse skin to UV radiation. PP1 catalytic subunit (PP1c) isoforms, PP1cα/γ and PP1cδ, are all localized to the cytoskeleton and cytosol of keratinocytes, but PP1cδ was found to be dominant over PP1α/γ in the nucleus. PP1c-silencing in HaCaT cells decreased the phosphatase activity and suppressed the viability of the cells. Exposure to a 10 J/cm(2) UVA dose induced HaCaT cell death and resulted in a 30% decrease of phosphatase activity. PP1c-silencing and UVA irradiation altered the gene expression profile of HaCaT cells and suggested that the expression of 19 genes was regulated by the combined treatments with many of these genes being involved in malignant transformation. Microarray analysis detected altered expression levels of genes coding for melanoma-associated proteins such as keratin 1/10, calcium binding protein S100A8 and histone 1b. Treatment of Balb/c mice with the PP1-specific inhibitor tautomycin (TM) exhibited increased levels of keratin 1/10 and S100A8, and a decreased level of histone 1b proteins following UVA irradiation. Moreover, TM treatment increased pigmentation of the skin which was even more apparent when TM was followed by UVA irradiation. Our data identify PP1 as a regulator of the normal homeostasis of keratinocytes and the UV-response.

Protein kinase Cδ promotes proliferation and induces malignant transformation in skeletal muscle.

In this paper, we investigated the isoform-specific roles of certain protein kinase C (PKC) isoforms in the regulation of skeletal muscle growth. Here, we provide the first intriguing functional evidence that nPKCδ (originally described as an inhibitor of proliferation in various cells types) is a key player in promoting both in vitro and in vivo skeletal muscle growth. Recombinant overexpression of a constitutively active nPKCδ in C2C12 myoblast increased proliferation and inhibited differentiation. Conversely, overexpression of kinase-negative mutant of nPKCδ (DN-nPKCδ) markedly inhibited cell growth. Moreover, overexpression of nPKCδ also stimulated in vivo tumour growth and induced malignant transformation in immunodeficient (SCID) mice whereas that of DN-nPKCδ suppressed tumour formation. The role of nPKCδ in the formation of rhabdomyosarcoma was also investigated where recombinant overexpression of nPKCδ in human rhabdomyosarcoma RD cells also increased cell proliferation and enhanced tumour formation in mouse xenografts. The other isoforms investigated (PKCα, ß, ε) exerted only minor (mostly growth-inhibitory) effects in skeletal muscle cells. Collectively, our data introduce nPKCδ as a novel growth-promoting molecule in skeletal muscles and invite further trials to exploit its therapeutic potential in the treatment of skeletal muscle malignancies.

Ankyrin domain of myosin 16 influences motor function and decreases protein phosphatase catalytic activity.

The unconventional myosin 16 (Myo16), which may have a role in regulation of cell cycle and cell proliferation, can be found in both the nucleus and the cytoplasm. It has a unique, eight ankyrin repeat containing pre-motor domain, the so-called ankyrin domain (My16Ank). Ankyrin repeats are present in several other proteins, e.g., in the regulatory subunit (MYPT1) of the myosin phosphatase holoenzyme, which binds to the protein phosphatase-1 catalytic subunit (PP1c). My16Ank shows sequence similarity to MYPT1. In this work, the interactions of recombinant and isolated My16Ank were examined in vitro. To test the effects of My16Ank on myosin motor function, we used skeletal muscle myosin or nonmuscle myosin 2B. The results showed that My16Ank bound to skeletal muscle myosin (K D ≈ 2.4 µM) and the actin-activated ATPase activity of heavy meromyosin (HMM) was increased in the presence of My16Ank, suggesting that the ankyrin domain can modulate myosin motor activity. My16Ank showed no direct interaction with either globular or filamentous actin. We found, using a surface plasmon resonance-based binding technique, that My16Ank bound to PP1cα (K D ≈ 540 nM) and also to PP1cδ (K D ≈ 600 nM) and decreased its phosphatase activity towards the phosphorylated myosin regulatory light chain. Our results suggest that one function of the ankyrin domain is probably to regulate the function of Myo16. It may influence the motor activity, and in complex with the PP1c isoforms, it can play an important role in the targeted dephosphorylation of certain, as yet unidentified, intracellular proteins.

Microcystin-LR, a cyanotoxin, modulates division of higher plant chloroplasts through protein phosphatase inhibition and affects cyanobacterial division.

Microcystin-LR (MC-LR) is a harmful cyanotoxin that inhibits 1 and 2A serine-threonine protein phosphatases. This study examines the influence of MC-LR on chloroplast division and the underlying mechanisms and consequences in Arabidopsis. MC-LR increased the frequency of dividing chloroplasts in hypocotyls in a time range of 1-96 h. At short-term exposures to MC-LR, small-sized chloroplasts (longitudinal diameters ≤6 µm) were more sensitive to these stimulatory effects, while both small and large chloroplasts showed stimulations at long-term exposure. After 48 h, the cyanotoxin increased the frequency of small-sized chloroplasts, indicating the stimulation of division. MC-LR inhibited protein phosphatases in whole hypocotyls and isolated chloroplasts, while it did not induce oxidative stress. We show for the first time that total cellular phosphatases play important roles in chloroplast division and that particular chloroplast phosphatases may be involved in these processes. Interestingly, MC-LR has a protective effect on cyanobacterial division during methyl-viologen (MV) treatments in Synechococcus PCC6301. MC-LR production has harmful effects on ecosystems and it may have an ancient cell division regulatory role in stressed cyanobacterial cells, the evolutionary ancestors of chloroplasts. We propose that cytoplasmic (eukaryotic) factors also contribute to the relevant effects of MC-LR in plants.

Protein phosphatase-1M and Rho-kinase affect exocytosis from cortical synaptosomes and influence neurotransmission at a glutamatergic giant synapse of the rat auditory system.

Protein phosphatase-1M (PP1M, myosin phosphatase) consists of a PP1 catalytic subunit (PP1c) and the myosin phosphatase target subunit-1 (MYPT1). RhoA-activated kinase (ROK) regulates PP1M via inhibitory phosphorylation of MYPT1. Using multidisciplinary approaches, we have studied the roles of PP1M and ROK in neurotransmission. Electron microscopy demonstrated the presence of MYPT1 and ROK in both pre- and post-synaptic terminals. Tautomycetin (TMC), a PP1-specific inhibitor, decreased the depolarization-induced exocytosis from cortical synaptosomes. trans-4-[(1R)-1-aminoethyl]-N-4-pyridinylcyclohexanecarboxamide dihydrochloride, a ROK-specific inhibitor, had the opposite effect. Mass spectrometry analysis identified several MYPT1-bound synaptosomal proteins, of which interactions of synapsin-I, syntaxin-1, calcineurin-A subunit, and Ca(2+) /calmodulin-dependent kinase II with MYPT1 were confirmed. In intact synaptosomes, TMC increased, whereas Y27632 decreased the phosphorylation levels of MYPT1(Thr696) , myosin-II light chain(Ser19) , synapsin-I(Ser9) , and syntaxin-1(Ser14) , indicating that PP1M and ROK influence their phosphorylation status. Confocal microscopy indicated that MYPT1 and ROK are present in the rat ventral cochlear nucleus both pre- and post-synaptically. Analysis of the neurotransmission in an auditory glutamatergic giant synapse demonstrated that PP1M and ROK affect neurotransmission via both pre- and post-synaptic mechanisms. Our data suggest that both PP1M and ROK influence synaptic transmission, but further studies are needed to give a full account of their mechanism of action.

Microcystin-LR, a protein phosphatase inhibitor, induces alterations in mitotic chromatin and microtubule organization leading to the formation of micronuclei in Vicia faba.

BACKGROUND AND AIMS: Microcystin-LR (MCY-LR) is a cyanobacterial toxin, a specific inhibitor of type 1 and 2A protein phosphatases (PP1 and PP2A) with significant impact on aquatic ecosystems. It has the potential to alter regulation of the plant cell cycle. The aim of this study was improved understanding of the mitotic alterations induced by cyanotoxin in Vicia faba, a model organism for plant cell biology studies. METHODS: Vicia faba seedlings were treated over the long and short term with MCY-LR purified in our laboratory. Short-term treatments were performed on root meristems synchronized with hydroxylurea. Sections of lateral root tips were labelled for chromatin, phosphorylated histone H3 and ß-tubulin via histochemical and immunohistochemical methods. Mitotic activity and the occurrence of mitotic alterations were detected and analysed by fluorescence microscopy. The phosphorylation state of histone H3 was studied by Western blotting. KEY RESULTS: Long-term MCY-LR exposure of lateral root tip meristems increased the percentage of either early or late mitosis in a concentration-dependent manner. We observed hypercondensed chromosomes and altered sister chromatid segregation (lagging chromosomes) leading to the formation of micronuclei, accompanied by the formation of disrupted, multipolar and monopolar spindles, disrupted phragmoplasts and the hyperphosphorylation of histone H3 at Ser10. Short-term MCY-LR treatment of synchronized cells showed that PP1 and PP2A inhibition delayed the onset of anaphase at 1 µg mL(-1) MCY-LR, accelerated cell cycle at 10 µg mL(-1) MCY-LR and induced the formation of lagging chromosomes. In this case mitotic microtubule alterations were not detected, but histone H3 was hyperphosphorylated. CONCLUSIONS: MCY-LR delayed metaphase-anaphase transition. Consequently, it induced aberrant chromatid segregation and micronucleus formation that could be associated with both H3 hyperphosphorylation and altered microtubule organization. However, these two phenomena seemed to be independent. The toxin may be a useful tool in the study of plant cell cycle regulation.

Epigallocatechine-3-gallate Inhibits the Adipogenesis of Human Mesenchymal Stem Cells via the Regulation of Protein Phosphatase-2A and Myosin Phosphatase.

Epigallocatechin-3-gallate (EGCG) has widespread effects on adipocyte development. However, the molecular mechanisms of EGCG are not fully understood. We investigate the adipogenic differentiation of human-derived mesenchymal stem cells, including lipid deposition and changes in the expression and phosphorylation of key transcription factors, myosin, protein phosphatase-2A (PP2A), and myosin phosphatase (MP). On day 6 of adipogenic differentiation, EGCG (1-20 µM) suppressed lipid droplet formation, which was counteracted by an EGCG-binding peptide for the 67 kDa laminin receptor (67LR), suggesting that EGCG acts via 67LR. EGCG decreased the phosphorylation of CCAAT-enhancer-binding protein beta via the activation of PP2A in a protein kinase A (PKA)-dependent manner, leading to the partial suppression of peroxisome proliferator-activated receptor gamma (PPARγ) and adiponectin expression. Differentiated cells exhibited a rounded shape, cortical actin filaments, and lipid accumulation. The EGCG treatment induced cell elongation, stress fiber formation, and less lipid accumulation. These effects were accompanied by the degradation of the MP target subunit-1 and increased the phosphorylation of the 20 kDa myosin light chain. Our results suggest that EGCG acts as an agonist of 67LR to inhibit adipogenesis via the activation of PP2A and suppression of MP. These events are coupled with the decreased phosphorylation and expression levels of adipogenic transcription factors and changes in cell shape, culminating in curtailed adipogenesis.

Affinity, avidity, and kinetics of target sequence binding to LC8 dynein light chain isoforms.

LC8 dynein light chain (DYNLL) is a highly conserved eukaryotic hub protein with dozens of binding partners and various functions beyond being a subunit of dynein and myosin Va motor proteins. Here, we compared the kinetic and thermodynamic parameters of binding of both mammalian isoforms, DYNLL1 and DYNLL2, to two putative consensus binding motifs (KXTQTX and XG(I/V)QVD) and report only subtle differences. Peptides containing either of the above motifs bind to DYNLL2 with micromolar affinity, whereas a myosin Va peptide (lacking the conserved Gln) and the noncanonical Pak1 peptide bind with K(d) values of 9 and 40 µM, respectively. Binding of the KXTQTX motif is enthalpy-driven, although that of all other peptides is both enthalpy- and entropy-driven. Moreover, the KXTQTX motif shows strikingly slower off-rate constant than the other motifs. As most DYNLL partners are homodimeric, we also assessed the binding of bivalent ligands to DYNLL2. Compared with monovalent ligands, a significant avidity effect was found as follows: K(d) values of 37 and 3.5 nM for a dimeric myosin Va fragment and a Leu zipper dimerized KXTQTX motif, respectively. Ligand binding kinetics of DYNLL can best be described by a conformational selection model consisting of a slow isomerization and a rapid binding step. We also studied the binding of the phosphomimetic S88E mutant of DYNLL2 to the dimeric myosin Va fragment, and we found a significantly lower apparent K(d) value (3 µM). We conclude that the thermodynamic and kinetic fine-tuning of binding of various ligands to DYNLL could have physiological relevance in its interaction network.

Transglutaminase 2 is needed for the formation of an efficient phagocyte portal in macrophages engulfing apoptotic cells.

Transglutaminase 2 (TG2), a protein cross-linking enzyme with many additional biological functions, acts as coreceptor for integrin beta(3). We have previously shown that TG2(-/-) mice develop an age-dependent autoimmunity due to defective in vivo clearance of apoptotic cells. Here we report that TG2 on the cell surface and in guanine nucleotide-bound form promotes phagocytosis. Besides being a binding partner for integrin beta(3), a receptor known to mediate the uptake of apoptotic cells via activating Rac1, we also show that TG2 binds MFG-E8 (milk fat globulin EGF factor 8), a protein known to bridge integrin beta(3) to apoptotic cells. Finally, we report that in wild-type macrophages one or two engulfing portals are formed during phagocytosis of apoptotic cells that are characterized by accumulation of integrin beta(3) and Rac1. In the absence of TG2, integrin beta(3) cannot properly recognize the apoptotic cells, is not accumulated in the phagocytic cup, and its signaling is impaired. As a result, the formation of the engulfing portals, as well as the portals formed, is much less efficient. We propose that TG2 has a novel function to stabilize efficient phagocytic portals.

Activation of Myosin Phosphatase by Epigallocatechin-Gallate Sensitizes THP-1 Leukemic Cells to Daunorubicin.

BACKGROUND: The Myosin Phosphatase (MP) holoenzyme is composed of a Protein Phosphatase type 1 (PP1) catalytic subunit and a regulatory subunit termed Myosin Phosphatase Target subunit 1 (MYPT1). Besides dephosphorylation of myosin, MP has been implicated in the control of cell proliferation via dephosphorylation and activation of the tumor suppressor gene products, retinoblastoma protein (pRb) and merlin. Inhibition of MP was shown to attenuate the drug-induced cell death of leukemic cells by chemotherapeutic agents, while activation of MP might have a sensitizing effect. OBJECTIVE: Recently, Epigallocatechin-Gallate (EGCG), a major component of green tea, was shown to activate MP by inducing the dephosphorylation of MYPT1 at phospho-Thr696 (MYPT1pT696), which might confer enhanced chemosensitivity to cancer cells. METHODS: THP-1 leukemic cells were treated with EGCG and Daunorubicin (DNR) and cell viability was analyzed. Phosphorylation of tumor suppressor proteins was detected by Western blotting. RESULTS: EGCG or DNR (at sub-lethal doses) alone had moderate effects on cell viability, while the combined treatment caused a significant decrease in the number of viable cells by enhancing apoptosis and decreasing proliferation. EGCG plus DNR decreased the phosphorylation level of MYPT1pT696, which was accompanied by prominent dephosphorylation of pRb. In addition, significant dephosphorylation of merlin was observed when EGCG and DNR were applied together. CONCLUSION: Our results suggest that EGCG-induced activation of MP might have a regulatory function in mediating the chemosensitivity of leukemic cells via dephosphorylation of tumor suppressor proteins.

Investigation of the Differences in Antithrombin to Heparin Binding among Antithrombin Budapest 3, Basel, and Padua Mutations by Biochemical and In Silico Methods.

Antithrombin (AT) is a serine protease inhibitor, its activity is highly accelerated by heparin. Mutations at the heparin-binding region lead to functional defect, type II heparin-binding site (IIHBS) AT deficiency. The aim of this study was to investigate and compare the molecular background of AT Budapest 3 (p.Leu131Phe, ATBp3), AT Basel (p.Pro73Leu), and AT Padua (p.Arg79His) mutations. Advanced in silico methods and heparin-binding studies of recombinant AT proteins using surface plasmon resonance method were used. Crossed immunoelectrophoresis and Differential Scanning Fluorimetry (NanoDSF) were performed in plasma samples. Heparin affinity of AT Padua was the lowest (KD = 1.08 × 10-6 M) and had the most severe consequences affecting the allosteric pathways of activation, moreover significant destabilizing effects on AT were also observed. KD values for AT Basel, ATBp3 and wild-type AT were 7.64 × 10-7 M, 2.15 × 10-8 M and 6.4 × 10-10 M, respectively. Heparin-binding of AT Basel was slower, however once the complex was formed the mutation had only minor effect on the secondary and tertiary structures. Allosteric activation of ATBp3 was altered, moreover decreased thermostability in ATBp3 homozygous plasma and increased fluctuations in multiple regions of ATBp3 were observed by in silico methods suggesting the presence of a quantitative component in the pathogenicity of this mutation due to molecular instability.

Microcystin-LR, a cyanobacterial toxin affects root development by changing levels of PIN proteins and auxin response in Arabidopsis roots.

Microcystin-LR (MCY-LR) is a heptapeptide toxin produced mainly by freshwater cyanobacteria. It strongly inhibits protein phosphatases PP2A and PP1. Functioning of the PIN family of auxin efflux carriers is crucial for plant ontogenesis and their functions depend on their reversible phosphorylation. We aimed to reveal the adverse effects of MCY-LR on PIN and auxin distribution in Arabidopsis roots and its consequences for root development. Relatively short-term (24 h) MCY-LR treatments decreased the levels of PIN1, PIN2 and PIN7, but not of PIN3 in tips of primary roots. In contrast, levels of PIN1 and PIN2 increased in emergent lateral roots and their levels depended on the type of PIN in lateral root primordia. DR5:GFP reporter activity showed that the cyanotoxin-induced decrease of auxin levels/responses in tips of main roots in parallel to PIN levels. Those alterations did not affect gravitropic response of roots. However, MCY-LR complemented the altered gravitropic response of crk5-1 mutants, defective in a protein kinase with essential role in the correct membrane localization of PIN2. For MCY-LR treated Col-0 plants, the number of lateral root primordia but not of emergent laterals increased and lateral root primordia showed early development. In conclusion, inhibition of protein phosphatase activities changed PIN and auxin levels, thus altered root development. Previous data on aquatic plants naturally co-occurring with the cyanotoxin showed similar alterations of root development. Thus, our results on the model plant Arabidopsis give a mechanistic explanation of MCY-LR phytotoxicity in aquatic ecosystems.