Mitophagy is a conserved intracellular catabolic process responsible for the selective removal of dysfunctional or superfluous mitochondria to maintain mitochondrial quality and need in cells. Here, ...we examine the mechanisms of receptor-mediated mitophagy activation, with the focus on BNIP3L/NIX mitophagy receptor, proven to be indispensable for selective removal of mitochondria during the terminal differentiation of reticulocytes. The molecular mechanisms of selecting damaged mitochondria from healthy ones are still very obscure. We investigated BNIP3L dimerization as a potentially novel molecular mechanism underlying BNIP3L-dependent mitophagy. Forming stable homodimers, BNIP3L recruits autophagosomes more robustly than its monomeric form. Amino acid substitutions of key transmembrane residues of BNIP3L, BNIP3L
G204A
or BNIP3L
G208V
, led to the abolishment of dimer formation, resulting in the lower LC3A-BNIP3L recognition and subsequently lower mitophagy induction. Moreover, we identified the serine 212 as the main amino acid residue at the C-terminal of BNIP3L, which extends to the intermembrane space, responsible for dimerization. In accordance, the phosphomimetic mutation BNIP3L
S212E
leads to a complete loss of BNIP3L dimerization. Thus, the interplay between BNIP3L phosphorylation and dimerization indicates that the combined mechanism of LIR phosphorylation and receptor dimerization is needed for proper BNIP3L-dependent mitophagy initiation and progression.
Abbreviations: AMBRA1: autophagy and beclin 1 regulator 1; Baf A1: bafilomycin A
1
; BH3: BCL2 homology 3; BNIP3: BCL2 interacting protein 3; BNIP3L/NIX: BCL2 interacting protein 3 like; CCCP: carbonyl cyanide 3-chlorophenylhydrazone; CoCl
2
: cobalt (II) chloride; FKBP8: FKBP prolyl isomerase 8; FUNDC1: FUN14 domain containing 1; GABARAP: GABA type A receptor-associated protein; GST: glutathione S-transferase; IMM: inner mitochondrial membrane; LIR: LC3-interacting region; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; OMM: outer mitochondrial membrane; PHB2: prohibitin 2; PI: propidium iodide; PINK1: PTEN induced kinase 1; TM: transmembrane domain; TOMM20: translocase of outer mitochondrial membrane 20
Extracellular nucleosides and nucleotides, such as adenosine and adenosine triphosphate (ATP), are involved in many physiological and pathological processes in adipose tissue (AT). It is becoming ...accepted that, in addition to the well-established sympathetic and hormonal system, purinergic receptors contribute significantly to regulation of adipocyte functions. Several receptor subtypes for both adenosine (P1) and ATP (P2X and P2Y) have been characterized in white adipocytes (WA) and brown adipocytes (BA). The effects mediated by adenosine and ATP on adipocytes are multiple and often differing, depending on specific receptors activated. Using a variety of agonists, antagonists and transgenic animals it has been demonstrated that adenosine and P2 receptors are involved in lipolysis, lipogenesis, adipokines secretion, glucose uptake, adipogenesis, cell proliferation, inflammation, and other processes. Given their central role in regulating many AT functions, purinergic receptors are considered potential therapeutic targets in different pathological conditions, such as obesity and type-2 diabetes. To achieve this goal, specific and potent P1 and P2 receptors activators and inhibitors are being developed and show promising results. However, more insight is needed into the function of P2 receptors in brown and beige adipocytes and their potential role in thermogenesis. This review aims at summarizing current knowledge on the patho-/physiological role of P1, P2X, and P2Y receptors in WA and BA and their potential exploitation for pharmacological intervention. Furthermore, we analyze impact of purinergic signaling in AT - in health and metabolic diseases.
Abstract This work reports, for the first time, a simple, rapid and sensitive square‐wave voltammetric method for the determination of ADB‐BUTINACA, which is currently one the most prevalent ...synthetic cannabinoids on the drug market in the European Union. The method is based on the direct electrochemical oxidation of ADB‐BUTINACA at a modification‐free boron‐doped diamond electrode in aqueous electrolyte solution. Studies indicated that the oxidation of ADB‐BUTINACA is pH‐independent, irreversible and controlled by diffusion. The best SWV response for ADB‐BUTINACA was obtained in 0.1 mol dm −3 KNO 3 buffered to pH 3, at a frequency of 100 Hz, a step potential of 2 mV and an amplitude of 50 mV. Under these conditions, the linear response for ADB‐BUTINACA was obtained in the concentration range of 1.1–104 μmol dm −3 , and calculated LOD and LOQ were 0.83 and 2.50 μmol dm −3 , respectively. The peak current based intra‐ and inter‐day precision values were 2.9 and 4.8 %, respectively. The spike‐recovery results in e‐liquid samples ranged between 92 and 104 %, confirming the applicability and good performance of the proposed method.
Mitophagy is a form of autophagy specialized to selectively remove mitochondria. Although the PINK1/Parkin pathway is the best described mitophagy of damaged mitochondria, receptor/mediated mitophagy ...seems to have a pivotal role in cellular development and specialization. The most studied mitophagy receptor BCL2/adenovirus E1B 19‐kDa‐interacting protein 3‐like (BNIP3L/NIX) is shown to be important for the programmed removal of healthy mitochondria during terminal differentiation of erythrocytes, but its role has been proven in various cell types. Despite recent advances in our understanding of its regulation by phosphorylation and dimerization, there remain numerous questions on how BNIP3L/NIX tightly balances between cellular life and death decisions. This brief review intends to summarize ongoing dilemmas related to BNIP3L/NIX.
The inactive form of the BCL2/adenovirus E1B 19‐kDa‐interacting protein 3‐like (BNIP3L/NIX) receptor is found as a monomer phosphorylated on C‐terminal serine 212. Upon mitophagy induction, the receptor is dephosphorylated and this allows BNIP3L/NIX monomers to form the active form of the receptor, BNIP3L/NIX dimer, which is also additionally activated with double phosphorylation near the LIR domain. Dimerization enables stronger recruitment of autophagosomes on selected mitochondria and more efficient mitophagy.
Aim
Extracellular ATP signalling is involved in many physiological and pathophysiological processes in several tissues, including adipose tissue. Adipocytes have crucial functions in lipid and ...glucose metabolism and they express purinergic receptors. However, the sources of extracellular ATP in adipose tissue are not well characterized. In the present study, we investigated the mechanism and regulation of ATP release in white adipocytes, and evaluated the role of extracellular ATP as potential autocrine and paracrine signal.
Methods
Online ATP release was monitored in C3H10T1/2 cells and freshly isolated murine adipocytes. The ATP release mechanism and its regulation were tested in cells exposed to adrenergic agonists, insulin, glucose load and pharmacological inhibitors. Cell metabolism was monitored using Seahorse respirometry and expression analysis of pannexin‐1 was performed on pre‐ and mature adipocytes. The ATP signalling was evaluated in live cell imaging (Ca2+, pore formation), glycerol release and its effect on macrophages was tested in co‐culture and migration assays.
Results
Here, we show that upon adrenergic stimulation white murine adipocytes release ATP through the pannexin‐1 pore that is regulated by a cAMP‐PKA–dependent pathway. The ATP release correlates with increased cell metabolism and is sensitive to glucose. Extracellular ATP induces Ca2+ signalling and lipolysis in adipocytes and promotes macrophage migration. Importantly, ATP release is markedly inhibited by insulin, which operates via the activation of phosphodiesterase 3.
Conclusions
Our findings reveal an insulin‐pannexin‐1‐purinergic signalling crosstalk in adipose tissue and we propose that deregulation of this signalling may contribute to adipose tissue inflammation and type 2 diabetes.
The purinergic signaling has an important role in regulating pancreatic exocrine secretion. The exocrine pancreas is also a site of one of the most serious cancer forms, the pancreatic ductal ...adenocarcinoma (PDAC). Here, we explore how the network of purinergic and adenosine receptors, as well as ecto-nucleotidases regulate normal pancreatic cells and various cells within the pancreatic tumor microenvironment. In particular, we focus on the P2X7 receptor, P2Y
and P2Y
receptors, as well as A
receptors and ecto-nucleotidases CD39 and CD73. Recent studies indicate that targeting one or more of these candidates could present new therapeutic approaches to treat pancreatic cancer. In pancreatic cancer, as much as possible of normal pancreatic function should be preserved, and therefore physiology of purinergic signaling in pancreas needs to be considered.
The electrochemical behaviour of capsaicin on a paraffin-impregnated graphite electrode (PIGE) was investigated using square-wave (SWV) and cyclic voltammetry (CV) in a wide range of pH values ...(2.0–11.0) and potential scan rates. The electrochemical oxidation of capsaicin is an irreversible process and occurs in a complex, pH dependent mechanism. In electrolytes with 2≤pH≤10 oxidation involves the transfer of two electrons and two protons, followed by chemical transformation and the formation of a product which undergoes a further reversible redox reaction. However, at pH close to pKa the product of two-electron electrochemical oxidation of capsaicin dissolves into the electrolyte.
Analysis of microparticles of dry ground pepper samples immobilized on the surface of PIGE was performed using SWV under optimal experimental conditions of pH 11, pulse amplitude of 50mV, frequency of 150Hz, and step potential of 2mV. The presence of capsaicin in samples was registered with good precision and sensitivity. The proposed methodology exploits the current intensity of capsaicinoids in pepper samples in order to classify samples according to their pungency. A strong positive correlation was observed between the measured peak currents for capsaicinoids in pepper samples and the average of Scoville heat units reported in the literature (R=91.3%).
BH3-only proteins integrate apoptosis and autophagy pathways, yet regulation and functional consequences of pathway cross-talk are not fully resolved. The BH3-only protein Bnip3 is an autophagy ...receptor that signals autophagic degradation of mitochondria (mitophagy) via interaction of its LC3-interacting region (LIR) with Atg8 proteins. Here we report that phosphorylation of serine residues 17 and 24 flanking the Bnip3 LIR promotes binding to specific Atg8 members LC3B and GATE-16. Using quantitative multispectral image-based flow cytometry, we demonstrate that enhancing Bnip3-Atg8 interactions via phosphorylation-mimicked LIR mutations increased mitochondrial sequestration, lysosomal delivery, and degradation. Importantly, mitochondria were targeted by mitophagy prior to cytochrome c release, resulting in reduced cellular cytochrome c release capacity. Intriguingly, pro-survival Bcl-xL positively regulated Bnip3 binding to LC3B, sequestration, and mitochondrial autophagy, further supporting an anti-apoptotic role for Bnip3-induced mitophagy. The ensemble of these results demonstrates that the phosphorylation state of the Bnip3 LIR signals either the induction of apoptosis or pro-survival mitophagy.
Background: Bnip3 is both a pro-apoptotic BH3-only protein and a mitochondrial autophagy receptor.
Results: Serine phosphorylation of the Bnip3 LC3-interacting region (LIR) increased binding to Atg8 members and consequently mitophagy, in a manner positively regulated by Bcl-xL.
Conclusion: The Bnip3 LIR activity state determines either pro-survival mitophagy or mitochondrial apoptosis.
Significance: Bnip3-induced mitophagy is serine kinase-regulated and thus a targetable pathway.
The mitophagy receptor Nix interacts with LC3/GABARAP proteins, targeting mitochondria into autophagosomes for degradation. Here we present evidence for phosphorylation-driven regulation of the ...Nix:LC3B interaction. Isothermal titration calorimetry and NMR indicate a ~100 fold enhanced affinity of the serine 34/35-phosphorylated Nix LC3-interacting region (LIR) to LC3B and formation of a very rigid complex compared to the non-phosphorylated sequence. Moreover, the crystal structure of LC3B in complex with the Nix LIR peptide containing glutamic acids as phosphomimetic residues and NMR experiments revealed that LIR phosphorylation stabilizes the Nix:LC3B complex via formation of two additional hydrogen bonds between phosphorylated serines of Nix LIR and Arg11, Lys49 and Lys51 in LC3B. Substitution of Lys51 to Ala in LC3B abrogates binding of a phosphomimetic Nix mutant. Functionally, serine 34/35 phosphorylation enhances autophagosome recruitment to mitochondria in HeLa cells. Together, this study provides cellular, biochemical and biophysical evidence that phosphorylation of the LIR domain of Nix enhances mitophagy receptor engagement.
Why and how do cells release ATP? It is not spilled energy. ATP becomes an extracellular regulator. Various cellular responses are initiated by purinergic receptors and signaling processes and are ...terminated by breakdown of ATP by ectonucleotidases. In epithelia, ATP regulates salt and water transport; other effects may be longer lasting.
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