A Lys–Trp Cation−π Interaction Mediates the Dimerization and Function of the Chloride Intracellular Channel Protein 1 Transmembrane Domain

133

110

The NLRP3 inflammasome is a multiprotein complex that regulates the activation of caspase-1 leading to the maturation of the proinflammatory cytokines IL-1β and IL-18, and promoting pyroptosis. Classically, the NLRP3 inflammasome in murine macrophages is activated by the recognition of pathogen-associated molecular patterns and by many structurally unrelated factors. Understanding the precise mechanism of NLRP3 activation by such wide array of stimuli remains elusive, but several signaling events, including cytosolic efflux and influx of select ions have been suggested. Accordingly, several studies have indicated a role of anion channels in NLRP3 inflammasome assembly, but their direct involvement has not been shown. Here, we report that the chloride intracellular channel proteins CLIC1 and CLIC4 participate in the regulation of the NLRP3 inflammasome. Confocal microscopy and cell fractionation experiments revealed that upon LPS stimulation of macrophages, CLIC1 and CLIC4 translocated into the nucleus and cellular membrane. In LPS/ATP-stimulated bone marrowderived macrophages (BMDMs), CLIC1 or CLIC4 siRNA transfection impaired transcription of IL-1β, ASC speck formation and secretion of mature IL-1β. Collectively, our results demonstrate that CLIC1 and CLIC4 participate both in the priming signal for IL-1b, and in NLRP3 activation.

Section: Introductionmentioning

confidence: 99%

The intracellular chloride channel proteins CLIC1 and CLIC4 induce IL-1β transcription and activate the NLRP3 inflammasome

Domingo-Fernandéz

Coll

Kearney

et al. 2017

133

110

“…In contrast, the polypeptides of MCA1 and MCA2 have a single transmembrane segment, as shown in this study, and assemble as a homotetramer to form a Ca 2ϩ -permeable channel (14,42). Chloride intracellular channels (CLICs) in mammals may have a single N-terminal transmembrane segment, but the number of subunits has not yet been established (43)(44)(45)(46). In this context, MCA1 and MCA2 are structurally unique among MS channels, and thus, elucidation of the molecular mechanisms of MCA1 and MCA2 involved in sensing mechanical stresses, including hypo-osmotic stress and membrane stretch, and in permeating Ca 2ϩ is important.…”

Section: Discussionmentioning

confidence: 79%

Transmembrane Topologies of Ca2+-permeable Mechanosensitive Channels MCA1 and MCA2 in Arabidopsis thaliana

Kamano

Kume

Iida

et al. 2015

Sensing mechanical stresses, including touch, stretch, compression, and gravity, is crucial for growth and development in plants. A good mechanosensor candidate is the Ca 2؉ -permeable mechanosensitive (MS) channel, the pore of which opens to permeate Ca 2؉ in response to mechanical stresses. However, the structure-function relationships of plant MS channels are poorly understood. Arabidopsis MCA1 and MCA2 form a homotetramer and exhibit Ca 2؉ -permeable MS channel activity; however, their structures have only been partially elucidated. The transmembrane topologies of these ion channels need to be determined in more detail to elucidate the underlying regulatory mechanisms. We herein determined the topologies of MCA1 and MCA2 using two independent methods, the Suc2C reporter and split-ubiquitin yeast two-hybrid methods, and found that both proteins are single-pass type I integral membrane proteins with extracellular N termini and intracellular C termini. These results imply that an EF hand-like motif, coiled-coil motif, and plac8 motif are all present in the cytoplasm. Thus, the activities of both channels can be regulated by intracellular Ca 2؉ and protein interactions.Mechanical stimuli affect plant growth, development, and resistance to herbivores (1-6). Natural stimuli, such as wind and gravity, modify the height and shape of grasses and trees.Touch retards the elongation of inflorescence and increases resistance to herbivores in Arabidopsis thaliana. Thus, stimulus perception is fundamental to plants, and the elucidation of mechano-sensors is important for understanding the molecular basis of plant mechanics and morphogenesis.A Ca 2ϩ -permeable mechanosensitive (MS) 8 channel has been suggested as a component of mechano-sensors (7-10). Using Nicotiana plumbaginifolia seedlings carrying the Ca 2ϩ -dependent photoprotein, aequorin, as an intracellular Ca 2ϩ indicator, a previous study reported that touch elicits an immediate increase in the cytosolic concentration of Ca 2ϩ ([Ca 2ϩ ] cyt ), which may act as a Ca 2ϩ signal (11). We recently identified novel Ca 2ϩ -permeable MS channels in Arabidopsis (A. thaliana), named MCA1 and MCA2 (mid1-complementing activity 1 and 2) (12-15). Both proteins share 74% identity in their amino acid sequences, form a homotetramer, have no homology to any known ion channels or transporters, and mediate Ca 2ϩ influx upon mechanical stimulation, such as hypo-osmotic shock and membrane stretch. Genes coding for MCA orthologs are found exclusively in the plant kingdom (9, 12). Rice and tobacco MCA proteins are also shown to mediate an increase in [Ca 2ϩ ] cyt upon hypo-osmotic shock (16,17).An in silico study suggested that MCA1 and MCA2 have several motifs, such as an EF hand-like motif, coiled-coil motif, and plac8 (DUF614) motif as well as a few predicted putative transmembrane segments (12, 13). To understand the molecular and physiological functions of both proteins and their motifs, their transmembrane topologies need to be elucidated in more detail. In the present study we...

“…Thereby, a cationformation in solution is characterized by a small red shift of a few nanometers, accompanied by an increase in fluorescence intensity of about 30 -40% (38,39). However, in contrast to spectra of proteins in solution, Peter et al (40) reported even small blue shifts for a peptide embedded in different membrane mimetics. Comparing the fluorescence spectra of the WT and the R735L mutant of yeast V-ATPase, we observed the expected intensity increase of ϳ30% (max of 339.81 Ϯ 1.40 nm for the WT and 341.66 Ϯ 2.03 nm for R735L) ( Fig.…”

Section: A Cation-interaction Facilitates Proton Transportmentioning

confidence: 99%

A cation–π interaction in a transmembrane helix of vacuolar ATPase retains the proton-transporting arginine in a hydrophobic environment

Hohlweg¹,

Wagner

Hofbauer

et al. 2018

Vacuolar ATPases are multisubunit protein complexes that are indispensable for acidification and pH homeostasis in a variety of physiological processes in all eukaryotic cells. An arginine residue (Arg-735) in transmembrane helix 7 (TM7) of subunit a of the yeast ATPase is known to be essential for proton translocation. However, the specific mechanism of its involvement in proton transport remains to be determined. Arginine residues are usually assumed to "snorkel" toward the protein surface when exposed to a hydrophobic environment. Here, using solution NMR spectroscopy, molecular dynamics simulations and in vivo yeast assays, we obtained evidence for the formation of a transient, membrane-embedded cation-π interaction in TM7 between Arg-735 and two highly conserved nearby aromatic residues, Tyr-733 and Trp-737. We propose a mechanism by which the transient, membrane-embedded cation-π complex provides the necessary energy to keep the charged side chain of Arg-735 within the hydrophobic membrane. Such cation-π interactions may define a general mechanism to retain charged amino acids in a hydrophobic membrane environment.