Inherited dysfunctional platelet P2Y12 receptor mutations associated with bleeding disorders

SummaryThe platelet adenosine 5’-diphosphate (ADP) receptor P2Y12 (P2Y12R) plays a critical role in platelet aggregation. The present report illustrates an update of dysfunctional platelet P2Y12R mutations diagnosed with congenital lifelong bleeding problems. Described patients with heterozygous or homozygous substitution in the P2Y12R gene and qualitative abnormalities of the platelet P2Y12R are summarized. Recently, a further dysfunctional variant of P2Y12R has been identified in two brothers who presented with a lifelong severe bleeding disorder. During in vitro aggregation studies, the patient´s platelets show a markedly reduced and rapid reversible ADP-promoted aggregation. A homozygous c.561T>A substitution that changes the codon for His187 to Gln (p.His187Gln) in the P2Y12R gene has been identified. This mutation causes no change in receptor expression but decreases the affinity of the ligand for the receptor, even at high concentrations. Structure modelling studies indicated that the p.His187Gln mutation, located in the fifth transmembrane spanning domain (TM5), impairs conformational changes of the receptor. Structural integrity of the TM5 region is necessary for agonist and antagonist binding and for correct receptor function.

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Regulation of interleukin 6 receptor expression in human monocytes and monocyte-derived macrophages. Comparison with the expression in human hepatocytes.

Journal of Experimental Medicine ◽

10.1084/jem.170.5.1537 ◽

1989 ◽

Vol 170 (5) ◽

pp. 1537-1549 ◽

Cited By ~ 84

Author(s):

J Bauer ◽

T M Bauer ◽

T Kalb ◽

T Taga ◽

G Lengyel ◽

...

Keyword(s):

Plasma Cells ◽

Human Peripheral Blood ◽

Receptor Expression ◽

Mrna Levels ◽

Blood Monocytes ◽

Inflammatory Conditions ◽

High Concentrations ◽

Human Primary Hepatocytes ◽

Stimulation Of

IL-6 is a cytokine with pleiotropic biological functions, including induction of the hepatic acute phase response and differentiation of activated B cells into Ig-secreting plasma cells. We found that human peripheral blood monocytes express the IL-6-R, which is undetectable on the large majority of lymphocytes of healthy individuals. Stimulation of monocytes by endotoxin or IL-1 causes a rapid downregulation of IL-6-R mRNA levels and a concomitant enhancement of IL-6 mRNA expression. IL-6 itself was found to suppress the IL-6-R at high concentrations. A gradual decrease of IL-6-R mRNA levels was observed along in vitro maturation of monocytes into macrophages. We show that downregulation of IL-6-R mRNA levels by IL-1 and IL-6 is monocyte specific, since IL-6-R expression is stimulated by both IL-1 and IL-6 in cultured human primary hepatocytes. Our data indicate that under noninflammatory conditions, monocytes may play a role in binding of trace amounts of circulating IL-6. Repression of monocytic IL-6-R and stimulation of hepatocytic IL-6-R synthesis may represent a shift of the IL-6 tissue targets under inflammatory conditions.

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IGF-I Regulates Pheochromocytoma Cell Proliferation and Survival In Vitro and In Vivo

Endocrinology ◽

10.1210/en.2012-1107 ◽

2012 ◽

Vol 153 (8) ◽

pp. 3724-3734 ◽

Cited By ~ 9

Author(s):

María Celia Fernández ◽

Marcela Venara ◽

Susana Nowicki ◽

Héctor E. Chemes ◽

Marta Barontini ◽

...

Keyword(s):

Critical Role ◽

Tumor Development ◽

Receptor Expression ◽

Vascular Density ◽

Receptor System ◽

Igf I ◽

Tumor Phenotype ◽

The Impact

IGFs are involved in malignant transformation and growth of several tissues, including the adrenal medulla. The present study was designed to evaluate the impact of IGF-I on pheochromocytoma development. We used a murine pheochromocytoma (MPC) cell line (MPC4/30) and an animal model with a reduction of 75% in circulating IGF-I levels [liver-IGF-I-deficient (LID) mice] to perform studies in vitro and in vivo. We found that, in culture, IGF-I stimulation increases proliferation, migration, and anchorage-independent growth, whereas it inhibits apoptosis of MPC cells. When injected to control and to LID mice, MPC cells grow and form tumors with features of pheochromocytoma. Six weeks after cell inoculation, all control mice developed sc tumors. In contrast, in 73% of LID mice, tumor development was delayed to 7–12 wk, and the remaining 27% did not develop tumors up to 12 wk after inoculation. LID mice harboring MPC cells and treated with recombinant human IGF-I (LID+) developed tumors as controls. Tumors developed in control, LID, and LID+ mice had similar histology and were similarly positive for IGF-I receptor expression. The apoptotic index was higher in tumors from LID mice compared with those from control mice, whereas vascular density was decreased. In summary, our work demonstrates that IGF-I has a critical role in maintaining tumor phenotype and survival of already transformed pheochromocytoma cells and is required for the initial establishment of these tumors, providing encouragement to carry on research studies to address the IGF-I/IGF-I receptor system as a target of therapeutic strategies for pheochromocytoma treatment in the future.

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Cryo-EM structure and in vitro DNA packaging of a thermophilic virus with supersized T=7 capsids

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1813204116 ◽

2019 ◽

Vol 116 (9) ◽

pp. 3556-3561 ◽

Cited By ~ 23

Author(s):

Oliver W. Bayfield ◽

Evgeny Klimuk ◽

Dennis C. Winkler ◽

Emma L. Hesketh ◽

Maria Chechik ◽

...

Keyword(s):

Conformational Changes ◽

Structural Integrity ◽

Thermus Thermophilus ◽

Dna Packaging ◽

Dna Viruses ◽

Portal Protein ◽

Single Vertex ◽

Double Stranded Dna ◽

Β Sheets

Double-stranded DNA viruses, including bacteriophages and herpesviruses, package their genomes into preformed capsids, using ATP-driven motors. Seeking to advance structural and mechanistic understanding, we established in vitro packaging for a thermostable bacteriophage, P23-45 of Thermus thermophilus. Both the unexpanded procapsid and the expanded mature capsid can package DNA in the presence of packaging ATPase over the 20 °C to 70 °C temperature range, with optimum activity at 50 °C to 65 °C. Cryo-EM reconstructions for the mature and immature capsids at 3.7-Å and 4.4-Å resolution, respectively, reveal conformational changes during capsid expansion. Capsomer interactions in the expanded capsid are reinforced by formation of intersubunit β-sheets with N-terminal segments of auxiliary protein trimers. Unexpectedly, the capsid has T=7 quasi-symmetry, despite the P23-45 genome being twice as large as those of known T=7 phages, in which the DNA is compacted to near-crystalline density. Our data explain this anomaly, showing how the canonical HK97 fold has adapted to double the volume of the capsid, while maintaining its structural integrity. Reconstructions of the procapsid and the expanded capsid defined the structure of the single vertex containing the portal protein. Together with a 1.95-Å resolution crystal structure of the portal protein and DNA packaging assays, these reconstructions indicate that capsid expansion affects the conformation of the portal protein, while still allowing DNA to be packaged. These observations suggest a mechanism by which structural events inside the capsid can be communicated to the outside.

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The antioxidant (−)-epigallocatechin-3-gallate inhibits activated hepatic stellate cell growth and suppresses acetaldehyde-induced gene expression

Biochemical Journal ◽

10.1042/bj20020894 ◽

2002 ◽

Vol 368 (3) ◽

pp. 695-704 ◽

Cited By ~ 66

Author(s):

Anping CHEN ◽

Li ZHANG ◽

Jianye XU ◽

Jun TANG

Keyword(s):

Transforming Growth Factor ◽

Stellate Cell ◽

Present Report ◽

Smooth Muscle Actin ◽

Critical Role ◽

Dependent Manner ◽

Liver Fibrogenesis ◽

Underlying Mechanisms

Activated hepatic stellate cells (HSC) are the primary source of excessive production of extracellular matrix during liver fibrogenesis. Although the underlying mechanisms remain incompletely understood, it is widely accepted that oxidative stress plays a critical role in liver fibrogenesis. Suppression of HSC growth and activation, as well as induction of apoptosis, have been proposed as therapeutic strategies for treatment and prevention of this disease. In the present report, we elucidated, for the first time, effects of the antioxidant (—)-epigallocatechin-3-gallate (EGCG), a major (and the most active) component of green tea extracts, on cultured HSC growth and activation. Our results revealed that EGCG significantly inhibited cultured HSC growth by inducing cell cycle arrest and apoptosis in a dose- and time-dependent manner. In addition, EGCG markedly suppressed the activation of cultured HSC as demonstrated by blocking transforming growth factor-β signal transduction and by inhibiting the expression of α1(I) collagen, fibronectin and α-smooth muscle actin genes induced by acetaldehyde, the most active metabolite of ethanol. Furthermore, EGCG reacted differently in the inhibition of nuclear factor-κB activity between cultured HSC with or without acetaldehyde stimulation. Taken together, our results indicated that EGCG was a novel and effective inhibitor for activated HSC growth and activation in vitro. Further studies are necessary to evaluate the effect of this polyphenol in prevention of quiescent HSC activation in vivo, and to further elucidate the underlying mechanisms.

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Phosphorylation and O-GlcNAcylation of the PHF-1 Epitope of Tau Protein Induce Local Conformational Changes of the C-Terminus and Modulate Tau Self-Assembly Into Fibrillar Aggregates

Frontiers in Molecular Neuroscience ◽

10.3389/fnmol.2021.661368 ◽

2021 ◽

Vol 14 ◽

Author(s):

François-Xavier Cantrelle ◽

Anne Loyens ◽

Xavier Trivelli ◽

Oliver Reimann ◽

Clément Despres ◽

...

Keyword(s):

Conformational Changes ◽

Tau Protein ◽

Posttranslational Modifications ◽

Self Assembly ◽

Critical Role ◽

Small Peptides ◽

C Terminus ◽

The Impact

Phosphorylation of the neuronal microtubule-associated Tau protein plays a critical role in the aggregation process leading to the formation of insoluble intraneuronal fibrils within Alzheimer’s disease (AD) brains. In recent years, other posttranslational modifications (PTMs) have been highlighted in the regulation of Tau (dys)functions. Among these PTMs, the O-β-linked N-acetylglucosaminylation (O-GlcNAcylation) modulates Tau phosphorylation and aggregation. We here focus on the role of the PHF-1 phospho-epitope of Tau C-terminal domain that is hyperphosphorylated in AD (at pS396/pS404) and encompasses S400 as the major O-GlcNAc site of Tau while two additional O-GlcNAc sites were found in the extreme C-terminus at S412 and S413. Using high resolution NMR spectroscopy, we showed that the O-GlcNAc glycosylation reduces phosphorylation of PHF-1 epitope by GSK3β alone or after priming by CDK2/cyclin A. Furthermore, investigations of the impact of PTMs on local conformation performed in small peptides highlight the role of S404 phosphorylation in inducing helical propensity in the region downstream pS404 that is exacerbated by other phosphorylations of PHF-1 epitope at S396 and S400, or O-GlcNAcylation of S400. Finally, the role of phosphorylation and O-GlcNAcylation of PHF-1 epitope was probed in in-vitro fibrillization assays in which O-GlcNAcylation slows down the rate of fibrillar assembly while GSK3β phosphorylation stimulates aggregation counteracting the effect of glycosylation.

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Biased localization of actin binding proteins by actin filament conformation

10.1101/2020.02.21.959791 ◽

2020 ◽

Author(s):

Andrew R Harris ◽

Pamela Jreij ◽

Brian Belardi ◽

Andreas Bausch ◽

Daniel A Fletcher

Keyword(s):

Actin Filament ◽

Single Molecule ◽

Conformational Changes ◽

Actin Filaments ◽

Binding Proteins ◽

Critical Role ◽

Actin Binding ◽

Cell Physiology ◽

Physical Constraints

ABSTRACTThe assembly of actin filaments into distinct cytoskeletal structures plays a critical role in cell physiology, but how proteins localize differentially to these structures within a shared cytoplasm remains unclear. Here, we show that the actin-binding domains of accessory proteins can be sensitive to filament conformational changes. Using a combination of live cell imaging and in vitro single molecule binding measurements, we show that tandem calponin homology domains (CH1-CH2) can be mutated to preferentially bind actin networks at the front or rear of motile cells, and we demonstrate that the affinity of CH1-CH2 domain mutants varies as actin filament conformation is altered by perturbations that include stabilizing drugs, physical constraints, and other binding proteins. These findings suggest that conformational heterogeneity of actin filaments in cells could help to direct accessory binding proteins to different actin cytoskeletal structures through a biophysical feedback loop.

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A role for platelet-derived growth factor β-receptor in a newborn rat model of endothelin-mediated pulmonary vascular remodeling

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00180.2004 ◽

2005 ◽

Vol 288 (6) ◽

pp. L1162-L1170 ◽

Cited By ~ 46

Author(s):

Robert P. Jankov ◽

Crystal Kantores ◽

Rosetta Belcastro ◽

Soojin Yi ◽

Ross A. Ridsdale ◽

...

Keyword(s):

Pulmonary Hypertension ◽

Smooth Muscle ◽

Rat Model ◽

Vascular Remodeling ◽

Critical Role ◽

Receptor Expression ◽

Newborn Rat ◽

Arterial Smooth Muscle

Newborn rats exposed to 60% O2 for 14 days develop endothelin (ET)-1-dependent pulmonary hypertension with vascular remodeling, characterized by increased smooth muscle cell (SMC) proliferation and medial thickening of pulmonary resistance arteries. Using immunohistochemistry and Western blot analyses, we examined the effect of exposure to 60% O2 on expression in the lung of receptors for the platelet-derived growth factors (PDGF), which are implicated in the pathogenesis of arterial smooth muscle hyperplasia. We observed a marked O2-induced upregulation of PDGF-α and -β receptors (PDGF-αR and -βR) on arterial smooth muscle. This led us to examine pulmonary vascular PDGF receptor expression in 60% O2-exposed rats given SB-217242, a combined ET receptor antagonist, which we found prevented the O2-induced upregulation of PDGF-βR, but not PDGF-αR, on arterial smooth muscle. PDGF-BB, a major PDGF-βR ligand, was found to be a potent in vitro inducer of hyperplasia and DNA synthesis in cultured pulmonary artery SMC from infant rats. A critical role for PDGF-βR ligands in arterial SMC proliferation was confirmed in vivo using a truncated soluble PDGF-βR intervention, which attenuated SMC proliferation induced by exposure to 60% O2. Collectively, these data are consistent with a major role for PDGF-βR-mediated SMC proliferation, acting downstream of increased ET-1 in a newborn rat model of 60% O2-induced pulmonary hypertension.

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Regulation and Function of Syk Tyrosine Kinase in Mast Cell Signaling and Beyond

Journal of Signal Transduction ◽

10.1155/2011/507291 ◽

2011 ◽

Vol 2011 ◽

pp. 1-9 ◽

Cited By ~ 27

Author(s):

Rodrigo Orlandini de Castro

Keyword(s):

Mast Cell ◽

Mast Cells ◽

Tyrosine Kinase ◽

Tyrosine Phosphorylation ◽

Conformational Changes ◽

Protein Tyrosine Kinase ◽

Critical Role ◽

Wild Type ◽

And Function

The protein tyrosine kinase Syk plays a critical role in FcεRI signaling in mast cells. Binding of Syk to phosphorylated immunoreceptor tyrosine-based activation motifs (p-ITAM) of the receptor subunits results in conformational changes and tyrosine phosphorylation at multiple sites that leads to activation of Syk. The phosphorylated tyrosines throughout the molecule play an important role in the regulation of Syk-mediated signaling. Reconstitution of receptor-mediated signaling in Syk-/- cells by wild-type Syk or mutants which have substitution of these tyrosines with phenylalanine together with in vitro assays has been useful strategies to understand the regulation and function of Syk.

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A blood–brain barrier overview on structure, function, impairment, and biomarkers of integrity

Fluids and Barriers of the CNS ◽

10.1186/s12987-020-00230-3 ◽

2020 ◽

Vol 17 (1) ◽

Author(s):

Hossam Kadry ◽

Behnam Noorani ◽

Luca Cucullo

Keyword(s):

Blood Brain Barrier ◽

Structural Integrity ◽

Critical Role ◽

Brain Barrier ◽

Clinical Aspects ◽

Advantages And Disadvantages ◽

Blood Brain ◽

The Brain

AbstractThe blood–brain barrier is playing a critical role in controlling the influx and efflux of biological substances essential for the brain’s metabolic activity as well as neuronal function. Thus, the functional and structural integrity of the BBB is pivotal to maintain the homeostasis of the brain microenvironment. The different cells and structures contributing to developing this barrier are summarized along with the different functions that BBB plays at the brain–blood interface. We also explained the role of shear stress in maintaining BBB integrity. Furthermore, we elaborated on the clinical aspects that correlate between BBB disruption and different neurological and pathological conditions. Finally, we discussed several biomarkers that can help to assess the BBB permeability and integrity in-vitro or in-vivo and briefly explain their advantages and disadvantages.

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Biased localization of actin binding proteins by actin filament conformation

Nature Communications ◽

10.1038/s41467-020-19768-9 ◽

2020 ◽

Vol 11 (1) ◽

Author(s):

Andrew R. Harris ◽

Pamela Jreij ◽

Brian Belardi ◽

Aaron M. Joffe ◽

Andreas R. Bausch ◽

...

Keyword(s):

Actin Filament ◽

Single Molecule ◽

Conformational Changes ◽

Actin Filaments ◽

Binding Proteins ◽

Critical Role ◽

Actin Binding ◽

Cell Physiology ◽

Binding Domains

AbstractThe assembly of actin filaments into distinct cytoskeletal structures plays a critical role in cell physiology, but how proteins localize differentially to these structures within a shared cytoplasm remains unclear. Here, we show that the actin-binding domains of accessory proteins can be sensitive to filament conformational changes. Using a combination of live cell imaging and in vitro single molecule binding measurements, we show that tandem calponin homology domains (CH1–CH2) can be mutated to preferentially bind actin networks at the front or rear of motile cells. We demonstrate that the binding kinetics of CH1–CH2 domain mutants varies as actin filament conformation is altered by perturbations that include stabilizing drugs and other binding proteins. These findings suggest that conformational changes of actin filaments in cells could help to direct accessory binding proteins to different actin cytoskeletal structures through a biophysical feedback loop.

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