The long noncoding RNA ADIPINT regulates human adipocyte metabolism via pyruvate carboxylase.

The pleiotropic function of long noncoding RNAs is well recognized, but their direct role in governing metabolic homeostasis is less understood. Here, we describe a human adipocyte-specific lncRNA, ADIPINT, that regulates pyruvate carboxylase, a pivotal enzyme in energy metabolism. We developed an approach, Targeted RNA-protein identification using Orthogonal Organic Phase Separation, which identifies that ADIPINT binds to pyruvate carboxylase and validated the interaction with electron microscopy. ADIPINT knockdown alters the interactome and decreases the abundance and enzymatic activity of pyruvate carboxylase in the mitochondria. Reduced ADIPINT or pyruvate carboxylase expression lowers adipocyte lipid synthesis, breakdown, and lipid content. In human white adipose tissue, ADIPINT expression is increased in obesity and linked to fat cell size, adipose insulin resistance, and pyruvate carboxylase activity. Thus, we identify ADIPINT as a regulator of lipid metabolism in human white adipocytes, which at least in part is mediated through its interaction with pyruvate carboxylase.

Optimizing purification of the peripheral membrane protein FAM92A1 fused to a modified spidroin tag.

Cryo-electron microscopy has revolutionized structural biology. In particular structures of proteins at the membrane interface have been a major contribution of cryoEM. Yet, visualization and characterization of peripheral membrane proteins remains challenging; mostly because there is no unified purification strategy for these proteins. FAM92A1 is a novel peripheral membrane protein that binds to the mitochondrial inner membrane. There, FAM92A1 dimers bind to the membrane and play an essential role in regulating the mitochondrial ultrastructure. Curiously, FAM92A1 has also an important function in ciliogenesis. FAM92A1 is part of the membrane bending Bin1/Amphiphsyin/RVS (BAR) domain protein family. Currently, there is no structure of FAM92A1, mostly because FAM92A1 is unstable and insoluble at high concentrations, like many BAR domain proteins. Yet, pure and concentrated protein is a necessity for screening to generate samples suitable for structure determination. Here, we present an optimized purification and expression strategy for dimeric FAM92A1. To our knowledge, we are the first to use the spidroin tag NT* to successfully purify a peripheral membrane protein. Our results show that NT* not only increases solubility but stabilizes FAM92A1 as a dimer. FAM92A1 fused to NT* is active because it is able to efficiently bend membranes. Taken together, our strategy indicates that this is a possible avenue to express and purify other challenging BAR domain proteins.

Coming of Age: Cryo-Electron Tomography as a Versatile Tool to Generate High-Resolution Structures at Cellular/Biological Interfaces.

Over the last few years, cryo electron microscopy has become the most important method in structural biology. While 80% of deposited maps are from single particle analysis, electron tomography has grown to become the second most important method. In particular sub-tomogram averaging has matured as a method, delivering structures between 2 and 5 Å from complexes in cells as well as in vitro complexes. While this resolution range is not standard, novel developments point toward a promising future. Here, we provide a guide for the workflow from sample to structure to gain insight into this emerging field.

CryoEM reveals BIN1 (isoform 8) does not bind to single actin filaments in vitro.

Cells change their appearance by a concerted action of the cytoskeleton and the plasma membrane. The machinery that bends the membrane includes Bin/Amphiphysin/Rvs (BAR) domain proteins. Recently BAR domain proteins garnered attention as actin regulators, either by recruiting actin regulating proteins or through binding to actin directly. BIN1 (an important protein in Alzheimer's Disease, heart disease and cancer) is one of the few BAR proteins that bind to actin directly. Here, we imaged a complex of BIN1 and actin with cryoEM. Our results reveal that BIN1 cannot be found on single actin filaments.

Cells Control BIN1-Mediated Membrane Tubulation by Altering the Membrane Charge.

The Bridging integrator 1 (BIN1)/Amphiphysin/Rvs (BAR) protein family is an essential part of the cell's machinery to bend membranes. BIN1 is a muscle-enriched BAR protein with an established role in muscle development and skeletal myopathies. Here, we demonstrate that BIN1, on its own, is able to form complex interconnected tubular systems in vitro, reminiscent of t-tubule system in muscle cells. We further describe how BIN1's electrostatic interactions regulate membrane bending: the ratio of negatively charged lipids in the bilayer altered membrane bending and binding properties of BIN1 and so did the manipulation of BIN1's surface charge. We show that the electrostatically mediated BIN1 membrane binding depended on the membrane curvature-it was less affected in liposomes with high curvature. Curiously, BIN1 membrane binding and bending was diminished in cells where the membrane's charge was experimentally reduced. Membrane bending was also reduced in BIN1 mutants where negative or positive charges in the BAR domain have been eliminated. This phenotype, characteristic of BIN1 mutants linked to myopathies, was rescued when the membrane charge was made more negative. The latter findings also show that cells can control tubulation at their membranes by simply altering the membrane charge and through it, the recruitment of BAR proteins and their interaction partners (e.g. dynamin).

The Pharmacological Heterogeneity of Nepenthone Analogs in Conferring Highly Selective and Potent κ-Opioid Agonistic Activities.

To develop novel analgesics with no side effects or less side effects than traditional opioids is highly demanded to treat opioid receptor mediated pain and addiction issues. Recently, κ-opioid receptor (KOR) has been established as an attractive target, although its selective agonists could bear heterogeneous pharmacological activities. In this study, we designed and synthesized two new series of nepenthone derivatives by inserting a spacer (carbonyl) between 6α,14α-endo-ethenylthebaine and the 7α-phenyl substitution of the skeleton and by substituting the 17-N-methyl group with a cyclopropylmethyl group. We performed in vitro tests (binding and functional assays) and molecular docking operations on our newly designed compounds. The results of wet-experimental measures and modeled binding structures demonstrate that these new compounds are selective KOR agonists with nanomolar level affinities. Compound 4 from these new derivatives showed the highest affinity (Ki = 0.4 ± 0.1 nM) and the highest selectivity (µ/κ = 339, δ/κ = 2034) toward KOR. The in vivo tests revealed that compound 4 is able to induce stronger (ED50 = 2.1 mg/kg) and much longer antinociceptive effect than that of the typical KOR agonist U50488H (ED50 = 4.4 mg/kg). Therefore, compound 4 can be used as a perfect lead compound for future design of potent analgesics acting through KOR.