Autophagy modulates dynamics of connexins at the plasma membrane in a ubiquitin-dependent manner

Different pathways contribute to the turnover of connexins, the main structural components of gap junctions (GJs). The cellular pool of connexins targeted to each pathway and the functional consequences of degradation through these degradative pathways are unknown. In this work, we focused on the contribution of macroautophagy to connexin degradation. Using pharmacological and genetic blockage of macroautophagy both in vitro and in vivo, we found that the cellular pool targeted by this autophagic system is primarily the one organized into GJs. Interruption of connexins' macroautophagy resulted in their retention at the plasma membrane in the form of functional GJs and subsequent increased GJ-mediated intercellular diffusion. Up-regulation of macroautophagy alone is not sufficient to induce connexin internalization and degradation. To better understand what factors determine the autophagic degradation of GJ connexins, we analyzed the changes undergone by the fraction of plasma membrane connexin 43 targeted for macroautophagy and the sequence of events that trigger this process. We found that Nedd4-mediated ubiquitinylation of the connexin molecule is required to recruit the adaptor protein Eps15 to the GJ and to initiate the autophagy-dependent internalization and degradation of connexin 43. This study reveals a novel regulatory role for macroautophagy in GJ function that is directly dependent on the ubiquitinylation of plasma membrane connexins.

Download Full-text

Di-Leucine Signals Mediate Targeting of Tyrosinase and Synaptotagmin to Synaptic-like Microvesicles within PC12 Cells

Molecular Biology of the Cell ◽

10.1091/mbc.10.11.3979 ◽

1999 ◽

Vol 10 (11) ◽

pp. 3979-3990 ◽

Cited By ~ 53

Author(s):

Anastasiya D. Blagoveshchenskaya ◽

Eric W. Hewitt ◽

Daniel F. Cutler

Keyword(s):

Plasma Membrane ◽

Pc12 Cells ◽

Brefeldin A ◽

Adaptor Protein ◽

Dependent Manner ◽

Protein Traffic ◽

C Terminus ◽

Targeting Signal

One pathway in forming synaptic-like microvesicles (SLMV) involves direct budding from the plasma membrane, requires adaptor protein 2 (AP2) and is brefeldin A (BFA) resistant. A second route leads from the plasma membrane to an endosomal intermediate from which SLMV bud in a BFA-sensitive, AP3-dependent manner. Because AP3 has been shown to bind to a di-leucine targeting signal in vitro, we have investigated whether this major class of targeting signals is capable of directing protein traffic to SLMV in vivo. We have found that a di-leucine signal within the cytoplasmic tail of human tyrosinase is responsible for the majority of the targeting of HRP-tyrosinase chimeras to SLMV in PC12 cells. Furthermore, we have discovered that a Met-Leu di-hydrophobic motif within the extreme C terminus of synaptotagmin I supports 20% of the SLMV targeting of a CD4-synaptotagmin chimera. All of the traffic to the SLMV mediated by either di-Leu or Met-Leu is BFA sensitive, strongly suggesting a role for AP3 and possibly for an endosomal intermediate in this process. The differential reduction in SLMV targeting for HRP-tyrosinase and CD4-synaptotagmin chimeras by di-alanine substitutions or BFA treatment implies that different proteins use the two routes to the SLMV to differing extents.

Download Full-text

A novel Netrin-1–sensitive mechanism promotes local SNARE-mediated exocytosis during axon branching

The Journal of Cell Biology ◽

10.1083/jcb.201311003 ◽

2014 ◽

Vol 205 (2) ◽

pp. 217-232 ◽

Cited By ~ 52

Author(s):

Cortney C. Winkle ◽

Leslie M. McClain ◽

Juli G. Valtschanoff ◽

Charles S. Park ◽

Christopher Maglione ◽

...

Keyword(s):

Plasma Membrane ◽

Cortical Neurons ◽

Direct Interaction ◽

Vesicle Fusion ◽

Dependent Manner ◽

Axon Branching ◽

Spatial Regulation ◽

Novel Model

Developmental axon branching dramatically increases synaptic capacity and neuronal surface area. Netrin-1 promotes branching and synaptogenesis, but the mechanism by which Netrin-1 stimulates plasma membrane expansion is unknown. We demonstrate that SNARE-mediated exocytosis is a prerequisite for axon branching and identify the E3 ubiquitin ligase TRIM9 as a critical catalytic link between Netrin-1 and exocytic SNARE machinery in murine cortical neurons. TRIM9 ligase activity promotes SNARE-mediated vesicle fusion and axon branching in a Netrin-dependent manner. We identified a direct interaction between TRIM9 and the Netrin-1 receptor DCC as well as a Netrin-1–sensitive interaction between TRIM9 and the SNARE component SNAP25. The interaction with SNAP25 negatively regulates SNARE-mediated exocytosis and axon branching in the absence of Netrin-1. Deletion of TRIM9 elevated exocytosis in vitro and increased axon branching in vitro and in vivo. Our data provide a novel model for the spatial regulation of axon branching by Netrin-1, in which localized plasma membrane expansion occurs via TRIM9-dependent regulation of SNARE-mediated vesicle fusion.

Download Full-text

Controversial Regulation of Gene Expression and Protein Transduction of Aquaporins under Drought and Salinity Stress

Plants ◽

10.3390/plants9121662 ◽

2020 ◽

Vol 9 (12) ◽

pp. 1662

Author(s):

Lucía Yepes-Molina ◽

Gloria Bárzana ◽

Micaela Carvajal

Keyword(s):

Plasma Membrane ◽

Messenger Rna ◽

Regulation Of Gene Expression ◽

Protein Transduction ◽

Mrna Synthesis ◽

Plasma Membrane Intrinsic Protein ◽

Entire Plant ◽

The One

Enhancement of the passage of water through membranes is one of the main mechanisms via which cells can maintain their homeostasis under stress conditions, and aquaporins are the main participants in this process. However, in the last few years, a number of studies have reported discrepancies between aquaporin messenger RNA (mRNA) expression and the number of aquaporin proteins synthesised in response to abiotic stress. These observations suggest the existence of post-transcriptional mechanisms which regulate plasma membrane intrinsic protein (PIP) trafficking to the plasma membrane. This indicates that the mRNA synthesis of some aquaporins could be modulated by the accumulation of the corresponding encoded protein, in relation to the turnover of the membranes. This aspect is discussed in terms of the results obtained: on the one hand, with isolated vesicles, in which the level of proteins present provides the membranes with important characteristics such as resistance and stability and, on the other, with isolated proteins reconstituted in artificial liposomes as an in vitro method to address the in vivo physiology of the entire plant.

Download Full-text

TRIM39 deficiency inhibits tumor progression and autophagic flux in colorectal cancer via suppressing the activity of Rab7

Cell Death and Disease ◽

10.1038/s41419-021-03670-3 ◽

2021 ◽

Vol 12 (4) ◽

Author(s):

Jia Hu ◽

Xueliang Ding ◽

Shaobo Tian ◽

Yanan Chu ◽

Zhibo Liu ◽

...

Keyword(s):

Colorectal Cancer ◽

Autophagic Flux ◽

Dependent Manner ◽

Functional Studies ◽

Normal Tissues ◽

Tumor Tissues ◽

Autophagic Degradation ◽

Activity Dependent

AbstractThe biological function of TRIM39, a member of TRIM family, remains largely unexplored in cancer, especially in colorectal cancer (CRC). In this study, we show that TRIM39 is upregulated in tumor tissues compared to adjacent normal tissues and associated with poor prognosis in CRC. Functional studies demonstrate that TRIM39 deficiency restrains CRC progression in vitro and in vivo. Our results further find that TRIM39 is a positive regulator of autophagosome–lysosome fusion. Mechanistically, TRIM39 interacts with Rab7 and promotes its activity via inhibiting its ubiquitination at lysine 191 residue. Depletion of TRIM39 inhibits CRC progression and autophagic flux in a Rab7 activity-dependent manner. Moreover, TRIM39 deficiency suppresses CRC progression through inhibiting autophagic degradation of p53. Thus, our findings uncover the roles as well as the relevant mechanisms of TRIM39 in CRC and establish a functional relationship between autophagy and CRC progression, which may provide promising approaches for the treatment of CRC.

Download Full-text

VEGFR2 induces c-Src signaling and vascular permeability in vivo via the adaptor protein TSAd

Journal of Experimental Medicine ◽

10.1084/jem.20111343 ◽

2012 ◽

Vol 209 (7) ◽

pp. 1363-1377 ◽

Cited By ~ 135

Author(s):

Zuyue Sun ◽

Xiujuan Li ◽

Sara Massena ◽

Simone Kutschera ◽

Narendra Padhan ◽

...

Keyword(s):

Vascular Permeability ◽

Adaptor Protein ◽

Cell Junctions ◽

Vegf Receptor ◽

Dependent Manner ◽

Src Tyrosine Kinase ◽

Rous Sarcoma ◽

Src Homology 2 Domain

Regulation of vascular endothelial (VE) growth factor (VEGF)–induced permeability is critical in physiological and pathological processes. We show that tyrosine phosphorylation of VEGF receptor 2 (VEGFR2) at Y951 facilitates binding of VEGFR2 to the Rous sarcoma (Src) homology 2-domain of T cell–specific adaptor (TSAd), which in turn regulates VEGF-induced activation of the c-Src tyrosine kinase and vascular permeability. c-Src was activated in vivo and in vitro in a VEGF/TSAd-dependent manner, and was regulated via increased phosphorylation at pY418 and reduced phosphorylation at pY527. Tsad silencing blocked VEGF-induced c-Src activation, but did not affect pathways involving phospholipase Cγ, extracellular regulated kinase, and endothelial nitric oxide. VEGF-induced rearrangement of VE–cadherin–positive junctions in endothelial cells isolated from mouse lungs, or in mouse cremaster vessels, was dependent on TSAd expression, and TSAd formed a complex with VE-cadherin, VEGFR2, and c-Src at endothelial junctions. Vessels in tsad−/− mice showed undisturbed flow and pressure, but impaired VEGF-induced permeability, as measured by extravasation of Evans blue, dextran, and microspheres in the skin and the trachea. Histamine-induced extravasation was not affected by TSAd deficiency. We conclude that TSAd is required for VEGF-induced, c-Src-mediated regulation of endothelial cell junctions and for vascular permeability.

Download Full-text

ABT-737 Triggers Caspase-Dependent Inhibition of Platelet Procoagulant Extracellular Vesicle Release during Apoptosis and Secondary Necrosis In Vitro

Thrombosis and Haemostasis ◽

10.1055/s-0039-1693694 ◽

2019 ◽

Vol 119 (10) ◽

pp. 1665-1674 ◽

Cited By ~ 4

Author(s):

Hao Wei ◽

Matthew T. Harper

Keyword(s):

Plasma Membrane ◽

Membrane Integrity ◽

Annexin V ◽

Extracellular Vesicle ◽

Ionophore A23187 ◽

Dependent Manner ◽

Plasma Membrane Integrity ◽

Secondary Necrosis

AbstractPlatelet lifespan is limited by activation of intrinsic apoptosis. Apoptotic platelets are rapidly cleared from the circulation in vivo. ABT-737 triggers platelet apoptosis and is a useful tool for studying this process. However, in vitro experiments lack clearance mechanisms for apoptotic platelets. To determine whether apoptotic platelets progress to secondary necrosis, apoptosis was triggered in human platelets with ABT-737, a BH3 mimetic. Platelet annexin V (AnV) binding, release of AnV+ extracellular vesicles (EVs), and loss of plasma membrane integrity were monitored by flow cytometry. ABT-737 triggered AnV binding, indicating phosphatidylserine exposure, release of AnV+ EVs, and a slow loss of plasma membrane integrity. The latter suggests that apoptotic platelets progress to secondary necrosis in vitro. These responses were dependent on caspase activation and Ca2+ entry. Surprisingly, although intracellular Ca2+ concentration increased, AnV+ EV release was not dependent on the Ca2+-dependent protease, calpain. On the contrary, ABT-737 downregulated the ability of the Ca2+ ionophore, A23187, to trigger calpain-dependent release of AnV+ EVs. This was dependent on caspase activity as, when caspases were inhibited, ABT-737 increased the ability of A23187 to trigger AnV+ EV release. These data suggest that apoptotic platelets progress to secondary necrosis unless they are cleared. This may affect the interpretation of ABT-737-triggered signaling in platelets in vitro. Ca2+-dependent AnV+ EV release is downregulated during apoptosis in a caspase-dependent manner, which may limit the potential consequences of secondary necrotic platelets.

Download Full-text

Rho-Kinase Phosphorylates COOH-terminal Threonines of Ezrin/Radixin/Moesin (ERM) Proteins and Regulates Their Head-to-Tail Association

The Journal of Cell Biology ◽

10.1083/jcb.140.3.647 ◽

1998 ◽

Vol 140 (3) ◽

pp. 647-657 ◽

Cited By ~ 600

Author(s):

Takeshi Matsui ◽

Masato Maeda ◽

Yoshinori Doi ◽

Shigenobu Yonemura ◽

Mutsuki Amano ◽

...

Keyword(s):

Plasma Membrane ◽

Actin Filament ◽

Catalytic Domain ◽

Rho Kinase ◽

Direct Interaction ◽

Dependent Manner ◽

Erm Proteins ◽

Filament Plasma

The ezrin/radixin/moesin (ERM) proteins are involved in actin filament/plasma membrane interaction that is regulated by Rho. We examined whether ERM proteins are directly phosphorylated by Rho- associated kinase (Rho-kinase), a direct target of Rho. Recombinant full-length and COOH-terminal half radixin were incubated with constitutively active catalytic domain of Rho-kinase, and ∼30 and ∼100% of these molecules, respectively, were phosphorylated mainly at the COOH-terminal threonine (T564). Next, to detect Rho-kinase–dependent phosphorylation of ERM proteins in vivo, we raised a mAb that recognized the T564-phosphorylated radixin as well as ezrin and moesin phosphorylated at the corresponding threonine residue (T567 and T558, respectively). Immunoblotting of serum-starved Swiss 3T3 cells with this mAb revealed that after LPA stimulation ERM proteins were rapidly phosphorylated at T567 (ezrin), T564 (radixin), and T558 (moesin) in a Rho-dependent manner and then dephosphorylated within 2 min. Furthermore, the T564 phosphorylation of recombinant COOH-terminal half radixin did not affect its ability to bind to actin filaments in vitro but significantly suppressed its direct interaction with the NH2-terminal half of radixin. These observations indicate that the Rho-kinase–dependent phosphorylation interferes with the intramolecular and/ or intermolecular head-to-tail association of ERM proteins, which is an important mechanism of regulation of their activity as actin filament/plasma membrane cross-linkers.

Download Full-text

Modulation of Erythrocyte Plasma Membrane Redox System Activity by Curcumin

Biochemistry Research International ◽

10.1155/2016/6025245 ◽

2016 ◽

Vol 2016 ◽

pp. 1-8 ◽

Cited By ~ 5

Author(s):

Prabhakar Singh ◽

Rajesh Kumar Kesharwani ◽

Krishna Misra ◽

Syed Ibrahim Rizvi

Keyword(s):

Plasma Membrane ◽

Curcuma Longa ◽

Docking Simulation ◽

Redox System ◽

Redox Status ◽

Dependent Manner ◽

Plasma Membrane Redox System ◽

Plasma Membrane Redox

Plasma membrane redox system (PMRS) is an electron transport chain system ubiquitously present throughout all cell types. It transfers electron from intracellular substrates to extracellular acceptors for regulation of redox status. Curcumin, isolated fromCurcuma longa,has modulatory effects on cellular physiology due to its membrane interaction ability and antioxidant potential. The present study investigates the effect of curcumin on PMRS activity of erythrocytes isolated from Wistar ratsin vitroandin vivoand validated through anin silicodocking simulation study using Molegro Virtual Docker (MVD). Effects of curcumin were also evaluated on level of glutathione (GSH) and the oxidant potential of plasma measured in terms of plasma ferric equivalent oxidative potentials (PFEOP). Results show that curcumin significantly (p<0.01) downregulated the PMRS activity in a dose-dependent manner. Molecular docking results suggest that curcumin interacts with amino acids at the active site cavity of cytochromeb5reductase, a key constituent of PMRS. Curcumin also increased the GSH level in erythrocytes and plasma while simultaneously decreasing the oxidant potential (PFEOP) of plasma. Altered PMRS activity and redox status are associated with the pathophysiology of several health complications including aging and diabetes; hence, the above finding may explain part of the role of curcumin in health beneficial effects.

Download Full-text

Interaction of amphiphysins with AP-1 clathrin adaptors at the membrane

Biochemical Journal ◽

10.1042/bj20121373 ◽

2013 ◽

Vol 450 (1) ◽

pp. 73-83 ◽

Cited By ~ 7

Author(s):

Sonja Huser ◽

Gregor Suri ◽

Pascal Crottet ◽

Martin Spiess

Keyword(s):

Plasma Membrane ◽

Brefeldin A ◽

Adaptor Protein ◽

Trans Golgi Network ◽

Src Homology 3 ◽

Amphiphysin 1 ◽

Src Homology ◽

Golgi Network

The assembly of clathrin/AP (adaptor protein)-1-coated vesicles on the trans-Golgi network and endosomes is much less studied than that of clathrin/AP-2 vesicles at the plasma membrane for endocytosis. In vitro, the association of AP-1 with protein-free liposomes had been shown to require phosphoinositides, Arf1 (ADP-ribosylation factor 1)–GTP and additional cytosolic factor(s). We have purified an active fraction from brain cytosol and found it to contain amphiphysin 1 and 2 and endophilin A1, three proteins known to be involved in the formation of AP-2/clathrin coats at the plasma membrane. Assays with bacterially expressed and purified proteins showed that AP-1 stabilization on liposomes depends on amphiphysin 2 or the amphiphysin 1/2 heterodimer. Activity is independent of the SH3 (Src homology 3) domain, but requires interaction of the WDLW motif with γ-adaptin. Endogenous amphiphysin in neurons and transfected protein in cell lines co-localize perinuclearly with AP-1 at the trans-Golgi network. This localization depends on interaction of clathrin and the adaptor sequence in the amphiphysins and is sensitive to brefeldin A, which inhibits Arf1-dependent AP-1 recruitment. Interaction between AP-1 and amphiphysin 1/2 in vivo was demonstrated by co-immunoprecipitation after cross-linking. These results suggest an involvement of amphiphysins not only with AP-2 at the plasma membrane, but also in AP-1/clathrin coat formation at the trans-Golgi network.

Download Full-text

Chickpea (Cicer arietinum L.) Lectin Exhibit Inhibition of ACE-I, α-amylase and α-glucosidase Activity

Protein and Peptide Letters ◽

10.2174/0929866526666190327130037 ◽

2019 ◽

Vol 26 (7) ◽

pp. 494-501 ◽

Cited By ~ 5

Author(s):

Sameer Suresh Bhagyawant ◽

Dakshita Tanaji Narvekar ◽

Neha Gupta ◽

Amita Bhadkaria ◽

Ajay Kumar Gautam ◽

...

Keyword(s):

Biological Effects ◽

Exchange Chromatography ◽

Health Concern ◽

Carbohydrate Binding ◽

Dependent Manner ◽

Concentration Dependent Manner ◽

Ic50 Values ◽

Chickpea Lectin

Background: Diabetes and hypertension are the major health concern and alleged to be of epidemic proportions. This has made it a numero uno subject at various levels of investigation. Glucosidase inhibitor provides the reasonable option in treatment of Diabetes Mellitus (DM) as it specifically targets post prandial hyperglycemia. The Angiotensin Converting Enzyme (ACE) plays an important role in hypertension. Therefore, inhibition of ACE in treatment of elevated blood pressure attracts special interest of the scientific community. Chickpea is a food legume and seeds contain carbohydrate binding protein- a lectin. Some of the biological properties of this lectin hitherto been elucidated. Methods: Purified by ion exchange chromatography, chickpea lectin was tested for its in vitro antioxidant, ACE-I inhibitory and anti-diabetic characteristic. Results: Lectin shows a characteristic improvement over the synthetic drugs like acarbose (oral anti-diabetic drug) and captopril (standard antihypertensive drug) when, their IC50 values are compared. Lectin significantly inhibited α-glucosidase and α-amylase in a concentration dependent manner with IC50 values of 85.41 ± 1.21 ҝg/ml and 65.05 ± 1.2 µg/ml compared to acarbose having IC50 70.20 ± 0.47 value of µg/ml and 50.52 ± 1.01 µg/ml respectively. β-Carotene bleaching assay showed antioxidant activity of lectin (72.3%) to be as active as Butylated Hydroxylanisole (BHA). In addition, lectin demonstrated inhibition against ACE-I with IC50 value of 57.43 ± 1.20 µg/ml compared to captopril. Conclusion: Lectin demonstrated its antioxidant character, ACE-I inhibition and significantly inhibitory for α-glucosidase and α-amylase seems to qualify as an anti-hyperglycemic therapeutic molecule. The biological effects of chickpea lectin display potential for reducing the parameters of medically debilitating conditions. These characteristics however needs to be established under in vivo systems too viz. animals through to humans.

Download Full-text