scholarly journals Endothelial-specific Crif1 deletion induces BBB maturation and disruption via the alteration of actin dynamics by impaired mitochondrial respiration

2020 ◽  
Vol 40 (7) ◽  
pp. 1546-1561
Author(s):  
Min Joung Lee ◽  
Yunseon Jang ◽  
Jeongsu Han ◽  
Soo J Kim ◽  
Xianshu Ju ◽  
...  
Keyword(s):  
Actin Cytoskeleton ◽  
Motor Behavior ◽  
Actin Dynamics ◽  
Toxic Substances ◽  
Atp Production ◽  
Junctional Proteins ◽  
Peripheral Immune Cells

Cerebral endothelial cells (ECs) require junctional proteins to maintain blood–brain barrier (BBB) integrity, restricting toxic substances and controlling peripheral immune cells with a higher concentration of mitochondria than ECs of peripheral capillaries. The mechanism underlying BBB disruption by defective mitochondrial oxidative phosphorylation (OxPhos) is unclear in a mitochondria-related gene-targeted animal model. To assess the role of EC mitochondrial OxPhos function in the maintenance of the BBB, we developed an EC-specific CR6-interactin factor1 ( Crif1) deletion mouse. We clearly observed defects in motor behavior, uncompacted myelin and leukocyte infiltration caused by BBB maturation and disruption in this mice. Furthermore, we investigated the alteration in the actin cytoskeleton, which interacts with junctional proteins to support BBB integrity. Loss of Crif1 led to reorganization of the actin cytoskeleton and a decrease in tight junction-associated protein expression through an ATP production defect in vitro and in vivo. Based on these results, we suggest that mitochondrial OxPhos is important for the maturation and maintenance of BBB integrity by supplying ATP to cerebral ECs.

scholarly journals The Role of Herbal Bioactive Components in Mitochondria Function and Cancer Therapy

2019 ◽  
Vol 2019 ◽  
pp. 1-12 ◽  
Author(s):  
Fangfang Tao ◽  
Yanrong Zhang ◽  
Zhiqian Zhang
Keyword(s):  
Cancer Therapy ◽  
Cancer Cell ◽  
Apoptotic Cell ◽  
Redox Status ◽  
Cancer Therapies ◽  
Bioactive Components ◽  
Atp Production

Mitochondria are highly dynamic double-membrane organelles which play a well-recognized role in ATP production, calcium homeostasis, oxidation-reduction (redox) status, apoptotic cell death, and inflammation. Dysfunction of mitochondria has long been observed in a number of human diseases, including cancer. Targeting mitochondria metabolism in tumors as a cancer therapeutic strategy has attracted much attention for researchers in recent years due to the essential role of mitochondria in cancer cell growth, apoptosis, and progression. On the other hand, a series of studies have indicated that traditional medicinal herbs, including traditional Chinese medicines (TCM), exert their potential anticancer effects as an effective adjunct treatment for alleviating the systemic side effects of conventional cancer therapies, for reducing the risk of recurrence and cancer mortality and for improving the quality of patients’ life. An amazing feature of these structurally diverse bioactive components is that majority of them target mitochondria to provoke cancer cell-specific death program. The aim of this review is to summarize the in vitro and in vivo studies about the role of these herbs, especially their bioactive compounds in the modulation of the disturbed mitochondrial function for cancer therapy.

Abstract MP247: Mitochondrial Complex V Is Regulated By GRK2 In The Heart

2021 ◽  
Vol 129 (Suppl_1) ◽  
Author(s):  
Kimberly Ferrero ◽  
Jessica M Pfleger ◽  
Kurt Chuprun ◽  
Eric Barr ◽  
Erhe Gao ◽  
...  
Keyword(s):  
Atp Synthase ◽  
Cardiac Injury ◽  
Atp Synthesis ◽  
Cardiac Metabolism ◽  
Neonatal Rat ◽  
Interacting Proteins ◽  
Atp Production

The GPCR kinase GRK2 is highly expressed the heart; importantly, during cardiac injury or heart failure (HF) both levels and activity of GRK2 increase. The role of GRK2 during HF is canonically studied upstream of β-adrenergic desensitization. However, GRK2 has a large interactome and noncanonical functions for this kinase are being uncovered. We have discovered that in the heart, GRK2 translocates to mitochondria ( mtGRK2 ) following injury and is associated with negative effects on cardiac metabolism. Thus, we have sought to identify the mechanism(s) by which GRK2 can regulate mitochondrial function. We hypothesize that mtGRK2 interacts with proteins which regulate bioenergetics and substrate utilization, and this never-before-described role may partially explain the altered mitochondrial phenotype seen following cardiac injury or HF. Stress-induced mitochondrial translocation of GRK2 was validated in neonatal rat ventricular myocytes, murine heart tissue and a cardiac-derived cell line. Consequently, the GRK2 interactome was mapped basally and under stress conditions in vitro, in vivo , and with tagged recombinant peptides. GRK2-interacting proteins were isolated via immunoprecipitation and analyzed via liquid chromatography-mass spectroscopy (LCMS). Proteomics analysis (IPA; Qiagen) identified mtGRK2 interacting proteins which were also involved in mitochondrial dysfunction. Excitingly, Complexes I, II, IV and V (ATP synthase) of the electron transport chain (ETC) were identified in the subset of mtGRK2-dysfunction partners. Several mtGRK2-ETC interactions were increased following stress, particularly those in Complex V. We further established that mtGRK2 phosphorylates some of the subunits of Complex V, particularly the ATP synthase barrel which is critical for ATP production in the heart. Specific amino acid residues on these subunits have been identified using PTM-LCMS and are currently being validated in a murine model of myocardial infarction. To support these data, we have also determined that alterations in either the levels or kinase activity of GRK2 appear to alter the enzymatic activity of Complex V in vitro , thus altering ATP production. In summary, the phosphorylation of the ATP synthesis machinery by mtGRK2 may be regulating some of the phenotypic effects of injured or failing hearts such as increased ROS production and reduced fatty acid metabolism. Research is ongoing in our lab to elucidate the novel role of GRK2 in regulating mitochondrial bioenergetics and cell death, thus uncovering an exciting, druggable novel target for rescuing cardiac function in patients with injured and/or failing hearts.

Force-dependent vinculin binding to talin in live cells: a crucial step in anchoring the actin cytoskeleton to focal adhesions

2014 ◽  
Vol 306 (6) ◽  
pp. C607-C620 ◽  
Author(s):  
Hiroaki Hirata ◽  
Hitoshi Tatsumi ◽  
Chwee Teck Lim ◽  
Masahiro Sokabe
Keyword(s):  
Actin Cytoskeleton ◽  
Focal Adhesions ◽  
Living Cells ◽  
Live Cells ◽  
Mechanical Forces ◽  
Actin Network ◽  
Crucial Step

Mechanical forces play a pivotal role in the regulation of focal adhesions (FAs) where the actin cytoskeleton is anchored to the extracellular matrix through integrin and a variety of linker proteins including talin and vinculin. The localization of vinculin at FAs depends on mechanical forces. While in vitro studies have demonstrated the force-induced increase in vinculin binding to talin, it remains unclear whether such a mechanism exists at FAs in vivo. In this study, using fibroblasts cultured on elastic silicone substrata, we have examined the role of forces in modulating talin-vinculin binding at FAs. Stretching the substrata caused vinculin accumulation at talin-containing FAs, and this accumulation was abrogated by expressing the talin-binding domain of vinculin (domain D1, which inhibits endogenous vinculin from binding to talin). These results indicate that mechanical forces loaded to FAs facilitate vinculin binding to talin at FAs. In cell-protruding regions, the actin network moved backward over talin-containing FAs in domain D1-expressing cells while it was anchored to FAs in control cells, suggesting that the force-dependent vinculin binding to talin is crucial for anchoring the actin cytoskeleton to FAs in living cells.

scholarly journals Mammalian Adenylyl Cyclase-associated Protein 1 (CAP1) Regulates Cofilin Function, the Actin Cytoskeleton, and Cell Adhesion

2013 ◽  
Vol 288 (29) ◽  
pp. 20966-20977 ◽  
Author(s):  
Haitao Zhang ◽  
Pooja Ghai ◽  
Huhehasi Wu ◽  
Changhui Wang ◽  
Jeffrey Field ◽  
...  
Keyword(s):  
Cell Adhesion ◽  
Actin Cytoskeleton ◽  
Hela Cells ◽  
Actin Polymerization ◽  
Actin Dynamics ◽  
Ras Signaling ◽  
Filamentous Actin ◽  
Camp Pathway

CAP (adenylyl cyclase-associated protein) was first identified in yeast as a protein that regulates both the actin cytoskeleton and the Ras/cAMP pathway. Although the role in Ras signaling does not extend beyond yeast, evidence supports that CAP regulates the actin cytoskeleton in all eukaryotes including mammals. In vitro actin polymerization assays show that both mammalian and yeast CAP homologues facilitate cofilin-driven actin filament turnover. We generated HeLa cells with stable CAP1 knockdown using RNA interference. Depletion of CAP1 led to larger cell size and remarkably developed lamellipodia as well as accumulation of filamentous actin (F-actin). Moreover, we found that CAP1 depletion also led to changes in cofilin phosphorylation and localization as well as activation of focal adhesion kinase (FAK) and enhanced cell spreading. CAP1 forms complexes with the adhesion molecules FAK and Talin, which likely underlie the cell adhesion phenotypes through inside-out activation of integrin signaling. CAP1-depleted HeLa cells also had substantially elevated cell motility as well as invasion through Matrigel. In summary, in addition to generating in vitro and in vivo evidence further establishing the role of mammalian CAP1 in actin dynamics, we identified a novel cellular function for CAP1 in regulating cell adhesion.

scholarly journals Ryanodine Receptor 1-mediated Ca2+ signaling and mitochondrial reprogramming modulate uterine serous cancer malignant phenotypes

2021 ◽  
Author(s):  
Li Zhang ◽  
Chunxian Huang ◽  
Tsz-Lun Yeung ◽  
Sammy Ferri-Borgogno ◽  
Chilam AuYeung ◽  
...  
Keyword(s):  
Molecular Mechanism ◽  
Ryanodine Receptor ◽  
Functional Role ◽  
Patient Survival ◽  
Uterine Cancer ◽  
Atp Production ◽  
Ryanodine Receptor 1

Abstract Background Uterine serous cancer (USC) is the most common non-endometrioid subtype of uterine cancer, and is also the most aggressive. Most patients will die of progressively chemotherapy-resistant disease, and the development of new therapies that can target USC remains a major unmet clinical need. This study sought to determine the molecular mechanism by which a novel unfavorable prognostic biomarker RYR1 identified in advanced USC confers their malignant phenotypes, and demonstrated the efficacy of targeting RYR1 by repositioned FDA-approved compounds in USC treatment. Methods TCGA USC dataset was analyzed to identify top genes that are associated with patient survival and can be targeted by FDA-approved compounds. The top gene RYR1 was selected and the functional role of RYR1 in USC progression was determined by silencing and over-expressing RYR1 in USC cells in vitro and in vivo. The molecular mechanism and signaling networks associated with the functional role of RYR1 in USC progression were determined by reverse phase protein arrays (RPPA), Western blot, and transcriptomic profiling analyses. The efficacy of the repositioned compound dantrolene on USC progression was determined using both in vitro and in vivo models. Results High expression level of ryanodine receptor 1 (RYR1) in the tumors is associated with shortened overall survival. Inhibition of RYR1 suppressed proliferation, migration and enhanced apoptosis through the Ca2+-dependent AKT/CREB/PGC-1α and AKT/HK1/2 signaling pathways, which modulate mitochondrial bioenergetics properties, including oxidative phosphorylation, ATP production, mitochondrial membrane potential, ROS production and TCA metabolites, and glycolytic activities in USC cells. Repositioned compound dantrolene suppressed USC progression in both in vitro and mouse models. Conclusions These findings provide insight into the mechanism by which RYR1 modulates the malignant phenotypes of USC and could aid in the development of dantrolene as a repurposed therapeutic agent for the treatment of USC to improve patient survival.

scholarly journals Role of Nicotinamide in DNA Damage, Mutagenesis, and DNA Repair

2010 ◽  
Vol 2010 ◽  
pp. 1-13 ◽  
Author(s):  
Devita Surjana ◽  
Gary M. Halliday ◽  
Diona L. Damian
Keyword(s):  
Dna Repair ◽  
Cellular Responses ◽  
Water Soluble ◽  
Vitamin B3 ◽  
Atp Production ◽  
Amide Form ◽  
Parp 1

Nicotinamide is a water-soluble amide form of niacin (nicotinic acid or vitamin B3). Both niacin and nicotinamide are widely available in plant and animal foods, and niacin can also be endogenously synthesized in the liver from dietary tryptophan. Nicotinamide is also commercially available in vitamin supplements and in a range of cosmetic, hair, and skin preparations. Nicotinamide is the primary precursor of nicotinamide adenine dinucleotide (NAD+), an essential coenzyme in ATP production and the sole substrate of the nuclear enzyme poly-ADP-ribose polymerase-1 (PARP-1). Numerousin vitroandin vivostudies have clearly shown that PARP-1 and NAD+status influence cellular responses to genotoxicity which can lead to mutagenesis and cancer formation. This paper will examine the role of nicotinamide in the protection from carcinogenesis, DNA repair, and maintenance of genomic stability.

scholarly journals Regulation of ARHGAP19 in the endometrial epithelium: a possible role in the establishment of uterine receptivity

2021 ◽  
Vol 19 (1) ◽  
Author(s):  
Jingjie Liang ◽  
Kui Li ◽  
Kaiyu Chen ◽  
Junyong Liang ◽  
Ti Qin ◽  
...  
Keyword(s):  
Rho Gtpase ◽  
Embryo Implantation ◽  
Luciferase Assay ◽  
Uterine Receptivity ◽  
Morphological Transformation ◽  
Upstream Regulator ◽  
Junctional Proteins

Abstract Background The establishment of uterine receptivity is essential for embryo implantation initiation and involves a significant morphological transformation in the endometrial epithelial cells (EECs). The remodeling of junctional complexes and membrane-associated cytoskeleton is crucial for epithelial transformation. However, little is known about how this process is regulated in EECs during the receptive phase. ARHGAP19 is a Rho GTPase-activating protein that participates in various cytoskeletal-related events, including epithelial morphogenesis. Here, we investigated the role of ARHGAP19 in endometrial epithelial transformation during the establishment of uterine receptivity. The upstream regulator of ARHGAP19 was also investigated. Methods ARHGAP19 expression was examined in mouse uteri during early pregnancy and in human EEC lines. The role of ARHGAP19 was investigated by manipulating its expression in EECs. The effect of ARHGAP19 on junctional proteins in EECs was examined by western blotting and immunofluorescence. The effect of ARHGAP19 on microvilli was examined by scanning electron microscopy. The upstream microRNA (miRNA) was predicted using online databases and validated by the dual-luciferase assay. The in vivo and in vitro effect of miRNA on endogenous ARHGAP19 was examined by uterine injection of miRNA agomirs and transfection of miRNA mimics or inhibitors. Results ARHGAP19 was upregulated in the receptive mouse uteri and human EECs. Overexpression of ARHGAP19 in non-receptive EECs downregulated the expression of junctional proteins and resulted in their redistribution. Meanwhile, upregulating ARHGAP19 reorganized the cytoskeletal structure of EECs, leading to a decline of microvilli and changes in cell configuration. These changes weakened epithelial cell polarity and promoted the transition of non-receptive EECs to a receptive phenotype. Besides, miR-192-5p, a miRNA that plays a key role in maintaining epithelial properties, was validated as an upstream regulator of ARHGAP19. Conclusion These results suggested that ARHGAP19 may contribute to the transition of EECs from a non-receptive to a receptive state by regulating the remodeling of junctional proteins and membrane-associated cytoskeleton.

scholarly journals Thymosin β4 is essential for thrombus formation by controlling the G-actin/F-actin equilibrium in platelets

Haematologica ◽  
2021 ◽  
Author(s):  
Inga Scheller ◽  
Sarah Beck ◽  
Vanessa Göb ◽  
Carina Gross ◽  
Raluca A. I. Neagoe ◽  
...  
Keyword(s):  
Thrombus Formation ◽  
Critical Role ◽  
Actin Dynamics ◽  
Thymosin Β4 ◽  
Clot Retraction ◽  
And Function ◽  
External Signals

Coordinated rearrangements of the actin cytoskeleton are pivotal for platelet biogenesis from megakaryocytes (MKs) but also orchestrate key functions of peripheral platelets in hemostasis and thrombosis, such as granule release, the formation of filopodia and lamellipodia, or clot retraction. Along with profilin (Pfn) 1, thymosin β4 (encoded by Tmsb4x) is one of the two main G-actin sequestering proteins within cells of higher eukaryotes, and its intracellular concentration is particularly high in cells that rapidly respond to external signals by increased motility, such as platelets. Here, we analyzed constitutive Tmsb4x knockout (KO) mice to investigate the functional role of the protein in platelet production and function. Thymosin β4 deficiency resulted in a macrothrombocytopenia with only mildly increased platelet volume and an unaltered platelet life span. MK numbers in the bone marrow (BM) and spleen were unaltered, however, Tmsb4x KO MKs showed defective proplatelet formation in vitro and in vivo. Thymosin β4 deficient platelets displayed markedly decreased G-actin levels and concomitantly increased F-actin levels resulting in accelerated spreading on fibrinogen and clot retraction. Moreover, Tmsb4x KO platelets showed activation defects and an impaired immunoreceptor tyrosine-based activation motif (ITAM) signaling downstream of the activating collagen receptor glycoprotein (GP) VI. These defects translated into impaired aggregate formation under flow, protection from occlusive arterial thrombus formation in vivo and increased tail bleeding times. In summary, these findings point to a critical role of thymosin β4 for actin dynamics during platelet biogenesis, platelet activation downstream of GPVI and thrombus stability.

Role of calcium in metabolic signaling between cardiac sarcoplasmic reticulum and mitochondria in vitro

2003 ◽  
Vol 284 (2) ◽  
pp. C285-C293 ◽  
Author(s):  
Robert S. Balaban ◽  
Salil Bose ◽  
Stephanie A. French ◽  
Paul R. Territo
Keyword(s):  
Energy Metabolism ◽  
Sarcoplasmic Reticulum ◽  
Atp Synthesis ◽  
Cardiac Sarcoplasmic Reticulum ◽  
Atp Production ◽  
Wide Range ◽  
Reconstituted System

The role of Ca2+ as a cytosolic signaling molecule between porcine cardiac sarcoplasmic reticulum (SR) ATPase and mitochondrial ATP production was evaluated in vitro. The Ca2+ sensitivity of these processes was determined individually and in a reconstituted system with SR and mitochondria in a 0.5:1 protein-to-cytochrome aa 3 ratio. The half-maximal concentration ( K 1/2) of SR ATPase was 335 nM Ca2+. The ATP synthesis dependence was similar with a K 1/2 of 243 nM for dehydrogenases and 114 nM for overall ATP production. In the reconstituted system, Ca2+ increased thapsigargin-sensitive ATP production (maximum ∼5-fold) with minimal changes in mitochondrial reduced nicotinamide adenine dinucleotide (NADH). NADH concentration remained stable despite graded increases in NADH turnover induced over a wide range of Ca2+ concentrations (0 to ∼500 nM). These data are consistent with a balanced activation of SR ATPase and mitochondrial ATP synthesis by Ca2+ that contributes to a homeostasis of energy metabolism metabolites. It is suggested that this balanced activation by cytosolic Ca2+ is partially responsible for the minimal alteration in energy metabolism intermediates that occurs with changes in cardiac workload in vivo.

scholarly journals Blockade of Intranigral and Systemic D3 Receptors Stimulates Motor Activity in the Rat Promoting a Reciprocal Interaction Among Glutamate, Dopamine, and GABA

Biomolecules ◽  
2019 ◽  
Vol 9 (10) ◽  
pp. 511 ◽  
Author(s):  
Rodríguez-Sánchez ◽  
Escartín-Pérez ◽  
Leyva-Gómez ◽  
Avalos-Fuentes ◽  
Paz-Bermúdez ◽  
...  
Keyword(s):  
Motor Activity ◽  
Motor Behavior ◽  
Sch 23390 ◽  
Ambulatory Activity ◽  
D3 Receptors ◽  
Dopamine D3 ◽  
In Vivo Administration

In vivo activation of dopamine D3 receptors (D3Rs) depresses motor activity. D3Rs are widely expressed in subthalamic, striatal, and dendritic dopaminergic inputs into the substantia nigra pars reticulata (SNr). In vitro studies showed that nigral D3Rs modulate their neurotransmitter release; thus, it could be that these changes in neurotransmitter levels modify the discharge of nigro-thalamic neurons and, therefore, motor behavior. To determine how the in vitro responses correspond to the in vivo responses, we examined the effect of intra-nigral and systemic blockade of D3Rs in the interstitial content of glutamate, dopamine, and GABA within the SNr using microdialysis coupled to motor activity determinations in freely moving rats. Intranigral unilateral blockade of D3R with GR 103,691 increased glutamate, dopamine, and GABA. Increments correlated with increased ambulatory distance, non-ambulatory activity, and induced contralateral turning. Concomitant blockade of D3R with D1R by perfusion of SCH 23390 reduced the increase of glutamate; prevented the increment of GABA, but not of dopamine; and abolished behavioral effects. Glutamate stimulates dopamine release by NMDA receptors, while blockade with kynurenic acid prevented the increase in dopamine and, in turn, of GABA and glutamate. Finally, systemic administration of D3R selective antagonist U 99194A increased glutamate, dopamine, and GABA in SNr and stimulated motor activity. Blockade of intra-nigral D1R with SCH 23390 prior to systemic U 99194A diminished increases in neurotransmitter levels and locomotor activity. These data highlight the pivotal role of presynaptic nigral D3 and D1R in the control of motor activity and help to explain part of the effects of the in vivo administration of D3R agents.

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