Kyungsoo Shin

Preferential apelin-13 production by the proprotein convertase PCSK3 is implicated in obesity

The peptide hormone apelin is translated as a 77‐residue preproprotein, truncated to the 55‐residue proapelin and, subsequently, to 13–36‐residue bioactive isoforms named apelin‐13 to ‐36. Proapelin is hypothesized to be cleaved to apelin‐36 and then to the shorter isoforms. However, neither the mechanism of proapelin processing nor the endoproteases involved have been determined. We show direct cleavage of proapelin to apelin‐13 by proprotein convertase subtilisin/kexin 3 (PCSK3, or furin) in vitro, with no production of longer isoforms. Conversely, neither PCSK1 nor PCSK7 has appreciable proapelin cleavage activity. Furthermore, we show that both proapelin and PCSK3 transcript expression levels are increased in adipose tissue with obesity and during adipogenesis, suggesting that PCSK3 is responsible for proapelin processing in adipose tissue.

Apela exhibits isoform- and headgroup-dependent modulation of micelle binding, peptide conformation and dynamics.

Apela (also referred to as ELABELA and toddler) is a peptide hormone that activates the apelin receptor (AR or APJ) to regulate cardiovascular system development and function. Here, we report the first biophysical characterization of three apela isoforms, apela-54, -32, and -11, alongside a monomeric C1S-apela-11 mutant, using circular dichroism (CD) spectropolarimetry and nuclear magnetic resonance (NMR) spectroscopy. The behaviour of apela-54 is consistent with a preprotein containing a hydrophobic, N-terminal signal peptide. The potential for apela-membrane binding, leading to membrane catalyzed interactions with AR, was tested comprehensively for apela-32 and -11 in the presence of membrane-mimetic dodecylphosphocholine (DPC), sodium dodecyl sulfate (SDS), and 1-palmitoyl-2-hydroxy-sn-glycero-3-[phospho-rac-(1-glycerol)] (LPPG) micelles. According to pulsed-field gradient diffusion NMR experiments, apela-32 interacts with all three micelles. Chemical shift perturbations indicate widespread interactions along apela, with DPC and LPPG micelles inducing short segments with α-helical character at distinct regions. Consistent with these data, ps-ns dynamics along the peptide backbone appear decreased in the presence of micelles. Apela-11 and C1S-apela-11, alternatively, interact preferentially with SDS and LPPG micelles, promoting β-turn character observable by CD. Distinct differences in membrane-interaction propensity are therefore apparent both as a function of apela isoform and of detergent headgroup. These results imply the potential for cell membrane involvement in apela-AR recognition and binding, with the implication that membrane catalysis has distinct functional and regulatory roles throughout the apelinergic system.

Bioactivity of the putative apelin proprotein expands the repertoire of apelin receptor ligands

BACKGROUND Apelin is a peptide ligand for a class A G-protein coupled receptor called the apelin receptor (AR or APJ) that regulates angiogenesis, the adipoinsular axis, and cardiovascular functions. Apelin has been shown to be bioactive as 13, 17, and 36 amino acid isoforms, C-terminal fragments of the putatively inactive 55-residue proprotein (proapelin or apelin-55). Although intracellular proprotein processing has been proposed, isolation of apelin-55 from colostrum and milk demonstrates potential for secretion prior to processing and the possibility of proapelin-AR interaction. METHODS Apelin isoform activity and potency were compared by an In-Cell Western™ assay for ERK phosphorylation using a stably AR-transfected HEK293A cell line. Conformational comparison of apelin isoforms was carried out by circular dichroism and heteronuclear solution-state nuclear magnetic resonance spectroscopy. RESULTS Apelin-55 is shown to activate the AR, with similar maximum ERK phophorylation response and potency to the shorter isoforms except for apelin-13, which exhibited a greater potency. Correlating to this shared activity, highly similar conformations are exhibited in all apelin isoforms for the shared C-terminal region responsible for receptor binding and activation. CONCLUSIONS AR activation by all apelin isoforms likely hinges upon shared conformation and dynamics in the C-terminus, with apelin-55 providing an alternative bioactive isoform despite the addition of 19N-terminal residues relative to apelin-36. GENERAL SIGNIFICANCE Beyond providing novel insight into the physiology of this system, re-annotation of proapelin to the bioactive apelin-55 isoform adds to the molecular toolkit for dissection of apelin-AR interactions and expands the repertoire of therapeutic targets for the apelinergic system.

Reovirus FAST Proteins Drive Pore Formation and Syncytiogenesis Using a Novel Helix-Loop-Helix Fusion-Inducing Lipid Packing Sensor

Pore formation is the most energy-demanding step during virus-induced membrane fusion, where high curvature of the fusion pore rim increases the spacing between lipid headgroups, exposing the hydrophobic interior of the membrane to water. How protein fusogens breach this thermodynamic barrier to pore formation is unclear. We identified a novel fusion-inducing lipid packing sensor (FLiPS) in the cytosolic endodomain of the baboon reovirus p15 fusion-associated small transmembrane (FAST) protein that is essential for pore formation during cell-cell fusion and syncytiogenesis. NMR spectroscopy and mutational studies indicate the dependence of this FLiPS on a hydrophobic helix-loop-helix structure. Biochemical and biophysical assays reveal the p15 FLiPS preferentially partitions into membranes with high positive curvature, and this partitioning is impeded by bis-ANS, a small molecule that inserts into hydrophobic defects in membranes. Most notably, the p15 FLiPS can be functionally replaced by heterologous amphipathic lipid packing sensors (ALPS) but not by other membrane-interactive amphipathic helices. Furthermore, a previously unrecognized amphipathic helix in the cytosolic domain of the reptilian reovirus p14 FAST protein can functionally replace the p15 FLiPS, and is itself replaceable by a heterologous ALPS motif. Anchored near the cytoplasmic leaflet by the FAST protein transmembrane domain, the FLiPS is perfectly positioned to insert into hydrophobic defects that begin to appear in the highly curved rim of nascent fusion pores, thereby lowering the energy barrier to stable pore formation.

Apelin conformational and binding equilibria upon micelle interaction primarily depend on membrane-mimetic headgroup

Apelin is one of two peptide hormones that activate the apelin receptor (AR or APJ) to regulate the cardiovascular system, central nervous system, and adipoinsular axis. Here, we apply circular dichroism (CD) spectropolarimetry and nuclear magnetic resonance (NMR) spectroscopy to characterize the potential membrane binding by the two longest bioactive apelin isoforms, apelin-55 and -36, using membrane-mimetic dodecylphosphocholine (DPC), sodium dodecyl sulfate (SDS), and 1-palmitoyl-2-hydroxy-sn-glycero-3-[phospho-rac-(1-glycerol)] (LPPG) micelles. Pulsed field gradient diffusion NMR experiments demonstrated preferential interaction of both apelin-55 and -36 with anionic SDS and LPPG micelles over zwitterionic DPC micelles. Chemical shift perturbations and changes in ps-ns scale dynamics of apelin-55 in all micelles were similarly localized along the polypeptide backbone, demonstrating clear dependence upon detergent headgroup, while comparison of chemical shifts between apelin-55 and apelin-36 showed negligible differences indicative of highly similar modes of micelle interaction. Notably, the observed behaviour was consistent with an ensemble averaged pair of free and bound states in fast exchange on the NMR timescale proportional to the fraction of micelle-bound protein, implying a similar conformational equilibrium regardless of headgroup and tailgroup. Membrane catalysis of apelin-AR binding would thus give rise to analogous behaviour in the essential C-terminal region common to all apelin isoforms.

Transmembrane Segment XI of the Na + /H + Antiporter of S. pombe is a Critical Part of the Ion Translocation Pore

The Na+/H+ exchanger of the plasma membrane of S. pombe (SpNHE1) removes intracellular sodium in exchange for an extracellular proton. We examined the structure and functional role of amino acids 360–393 of putative transmembrane (TM) segment XI of SpNHE1. Structural analysis suggested that it had a helical propensity over amino acids 360–368, an extended region from 369–378 and was helical over amino acids 379–386. TM XI was sensitive to side chain alterations. Mutation of eight amino acids to alanine resulted in loss of one or both of LiCl or NaCl tolerance when re-introduced into SpNHE1 deficient S. pombe. Mutation of seven other amino acids had minor effects. Analysis of structure and functional mutations suggested that Glu361 may be involved in cation coordination on the cytoplasmic face of the protein with a negative charge in this position being important. His367, Ile371 and Gly372 were important in function. Ile371 may have important hydrophobic interactions with other residues and Gly372 may be important in maintaining an extended conformation. Several residues from Val377 to Leu384 are important in function possibly involved in hydrophobic interactions with other amino acids. We suggest that TM XI forms part of the ion translocation core of this Na+/H+ exchanger.

Mixed Fluorotryptophan Substitutions at the Same Residue Expand the Versatility of 19F Protein NMR Spectroscopy

The strategy of applying fluorine NMR to characterize ligand binding to a membrane protein prepared with mixtures of tryptophans substituted with F at different positions on the indole ring was tested. The 19 F NMR behavior of 4-, 5-, 6-, and 7-fluorotryptophan were directly compared as a function of both micellar environment and fragment size for two overlapping apelin receptor (AR/APJ) segments; one with a single transmembrane (TM) helix and two tryptophan residues, the other with three TM helices and two additional tryptophan residues. Chemical shifts, peak patterns, and nuclear spin relaxation rates were observed to vary as a function of micellar conditions and F substitution position in the indole ring, with the exposure of a given residue to micelle or solvent being the primary differentiating factor. Titration of the 3-TM AR segment biosynthetically prepared as a mixture of 5- and 7-fluorotryptophan-containing isoforms by two distinct peptide ligands (apelin-36 and apela-32) demonstrated site-specific 19 F peak intensity changes for one ligand but not the other. In contrast, both ligands perturbed 1 H-15 N HSQC peak patterns to a similar degree. Characterization of multiple fluorotryptophan types for a given set of tryptophan residues, thus, significantly augments the potential to apply 19 F NMR to track otherwise obscure modulation of protein conformation and dynamics without an explicit requirement for mutagenesis or chemical modification.