Interaction modes of human orexin 2 receptor with selective and nonselective antagonists studied by NMR spectroscopy.

Orexin neuropeptides have many physiological roles in the sleep-wake cycle, feeding behavior, reward demands, and stress responses by activating cognitive receptors, the orexin receptors (OX1R and OX2R), distributed in the brain. There are only subtle differences between OX1R and OX2R in the orthosteric site, which has hindered the rational development of subtype-selective antagonists. In this study, we utilized solution-state NMR to capture the structural plasticity of OX2R labeled with CH-ε-methionine in complex with antagonists.

Characterizing conformational ensembles of multi-domain proteins using anisotropic paramagnetic NMR restraints.

It has been over two decades since paramagnetic NMR started to form part of the essential techniques for structural analysis of proteins under physiological conditions. Paramagnetic NMR has significantly expanded our understanding of the inherent flexibility of proteins, in particular, those that are formed by combinations of two or more domains. Here, we present a brief overview of techniques to characterize conformational ensembles of such multi-domain proteins using paramagnetic NMR restraints produced through anisotropic metals, with a focus on the basics of anisotropic paramagnetic effects, the general procedures of conformational ensemble reconstruction, and some representative reweighting approaches.

Conformational Space Sampled by Domain Reorientation of Linear Diubiquitin Reflected in Its Binding Mode for Target Proteins.

Linear polyubiquitin chains regulate diverse signaling proteins, in which the chains adopt various conformations to recognize different target proteins. Thus, the structural plasticity of the chains plays an important role in controlling the binding events. Herein, paramagnetic NMR spectroscopy is employed to explore the conformational space sampled by linear diubiquitin, a minimal unit of linear polyubiquitin, in its free state.

Tracking the 3D Rotational Dynamics in Nanoscopic Biological Systems.

The rotation of an object cannot be fully tracked without understanding a set of three angles, namely, roll, pitch, and yaw. Tracking these angles as a three-degrees-of-freedom (3-DoF) rotation is a fundamental measurement, facilitating, for example, attitude control of a ship, image stabilization to reduce camera shake, and self-driving cars. Until now, however, there has been no method to track 3-DoF rotation to measure nanometer-scale dynamics in biomolecules and live cells.

An innate interaction between IL-18 and the propeptide that inactivates its precursor form.

Uncontrolled secretion of mature interleukin (IL)-1β and IL-18 is responsible for severe autoinflammatory or autoimmune disorders and various allergic diseases. Here we report an intramolecular interaction between IL-18 and its propeptide, which is proteolytically removed from its precursor proIL-18 during maturation. The intramolecular interaction was recapitulated intermolecularly using recombinant propeptide.

Intramolecular interaction suggests an autosuppression mechanism for the innate immune adaptor protein MyD88.

MyD88 (myeloid differentiation factor 88) is an important protein in innate immunity. Two structural domains of MyD88 have been well characterized separately, but the global architecture of full-length MyD88 remained unclear. Here, we propose an autosuppressive mechanism of MyD88 regulated by the intramolecular interaction between the two domains.

Cooperative Domain Formation by Homologous Motifs in HOIL-1L and SHARPIN Plays A Crucial Role in LUBAC Stabilization.

The linear ubiquitin chain assembly complex (LUBAC) participates in inflammatory and oncogenic signaling by conjugating linear ubiquitin chains to target proteins. LUBAC consists of the catalytic HOIP subunit and two accessory subunits, HOIL-1L and SHARPIN. Interactions between the ubiquitin-associated (UBA) domains of HOIP and the ubiquitin-like (UBL) domains of two accessory subunits are involved in LUBAC stabilization, but the precise molecular mechanisms underlying the formation of stable trimeric LUBAC remain elusive.

Hexaphyrin as a Potential Theranostic Dye for Photothermal Therapy and F Magnetic Resonance Imaging.

Two features of meso-Aryl-substituted expanded porphyrins suggest suitability as theranostic agents. They have excellent absorption in near infrared (NIR) region, and they offer the possibility of introduction of multiple fluorine atoms at structurally equivalent positions. Here, hexaphyrin (hexa) was synthesized from 2,6-bis(trifluoromethyl)-4-formyl benzoate and pyrrole and evaluated as a novel expanded porphyrin with the above features.

UCP3 is associated with Hax-1 in mitochondria in the presence of calcium ion.

Uncoupling protein 3 (UCP3) is known to regulate energy dissipation, proton leakage, fatty acid oxidation, and oxidative stress. To identify the putative protein regulators of UCP3, we performed yeast two-hybrid screens. Here we report that UCP3 interacted with HS-1 associated protein X-1 (Hax-1), an anti-apoptotic protein that was localized in the mitochondria, and is involved in cellular responses to Ca(2+).

Structural analysis of the TKB domain of ubiquitin ligase Cbl-b complexed with its small inhibitory peptide, Cblin.

Cbl-b is a RING-type ubiquitin ligase. Previously, we showed that Cbl-b-mediated ubiquitination and proteosomal degradation of IRS-1 contribute to muscle atrophy caused by unloading stress. The phospho-pentapeptide DGpYMP (Cblin) mimics Tyr612-phosphorylated IRS-1 and inhibits the Cbl-b-mediated ubiquitination and degradation of IRS-1 in vitro and in vivo.

Selective Labeling of Proteins on Living Cell Membranes Using Fluorescent Nanodiamond Probes.

The impeccable photostability of fluorescent nanodiamonds (FNDs) is an ideal property for use in fluorescence imaging of proteins in living cells. However, such an application requires highly specific labeling of the target proteins with FNDs. Furthermore, the surface of unmodified FNDs tends to adsorb biomolecules nonspecifically, which hinders the reliable targeting of proteins with FNDs.

Selective autophagic receptor p62 regulates the abundance of transcriptional coregulator ARIP4 during nutrient starvation.

Transcriptional coregulators contribute to several processes involving nuclear receptor transcriptional regulation. The transcriptional coregulator androgen receptor-interacting protein 4 (ARIP4) interacts with nuclear receptors and regulates their transcriptional activity. In this study, we identified p62 as a major interacting protein partner for ARIP4 in the nucleus.

Optically Detected Magnetic Resonance of Nanodiamonds In Vivo; Implementation of Selective Imaging and Fast Sampling.

Single-molecule fluorescence measurements of biological samples frequently suffer from background autofluorescence originating from fluorescent materials pre-existing in living samples, and from unstable photo-physical properties of fluorescent labeling molecules. In this study, we first describe our method of selective imaging of nanodiamonds containing nitrogen-vacancy centers, promising fluorescent color centers, by a combination of optically detected magnetic resonance. The resultant images exhibit perfect elimination of extraneous fluorescence in real-time microscope observations.

[Non-invasive analysis of proteins in living cells using NMR spectroscopy].

NMR spectroscopy enables structural analyses of proteins and has been widely used in the structural biology field in recent decades. NMR spectroscopy can be applied to proteins inside living cells, allowing characterization of their structures and dynamics in intracellular environments. The simplest "in-cell NMR" approach employs bacterial cells; in this approach, live Escherichia coli cells overexpressing a specific protein are subjected to NMR.

The unexpected role of polyubiquitin chains in the formation of fibrillar aggregates.

Ubiquitin is known to be one of the most soluble and stably folded intracellular proteins, but it is often found in inclusion bodies associated with various diseases including neurodegenerative disorders and cancer. To gain insight into this contradictory behaviour, we have examined the physicochemical properties of ubiquitin and its polymeric chains that lead to aggregate formation. We find that the folding stability of ubiquitin chains unexpectedly decreases with increasing chain length, resulting in the formation of amyloid-like fibrils.

Magnetic resonance imaging of tumor with a self-traceable phosphorylcholine polymer.

Polymers are concentration-amplified with respect to the monomeric units. We show here that a phosphorylcholine polymer enriched with (13)C/(15)N at the methyl groups is self-traceable by multiple-resonance (heteronuclear-correlation) NMR in tumor-bearing mice inoculated with the mouse rectal cancer cell line (colon 26). Preliminary measurements indicated that the present polymeric nanoprobe was satisfactorily distinguished from lipids and detectable with far sub-micromolar spectroscopic and far sub-millimolar imaging sensitivities.

The structural basis for receptor recognition of human interleukin-18.

Interleukin (IL)-18 is a proinflammatory cytokine that belongs to the IL-1 family and plays an important role in inflammation. The uncontrolled release of this cytokine is associated with severe chronic inflammatory disease. IL-18 forms a signalling complex with the IL-18 receptor α (Rα) and β (Rβ) chains at the plasma membrane, which induces multiple inflammatory cytokines.

Purification, crystallization and preliminary X-ray crystallographic analysis of human IL-18 and its extracellular complexes.

Interleukin-18 (IL-18), a pro-inflammatory cytokine belonging to the interleukin-1 (IL-1) family, is involved in the pathogenesis of autoimmune/autoinflammatory and allergic diseases such as juvenile idiopathic arthritis and bronchial asthma. IL-18 forms a signalling complex with the IL-18 receptor α (IL-18Rα) and β (IL-18Rβ) chains; however, the detailed activation mechanism remains unclear. Here, the IL-18-IL-18Rα binary and IL-18-IL-18Rα-IL-18Rβ ternary complexes were purified and crystallized as well as IL-18 alone.

Solution structure of the ubiquitin-associated (UBA) domain of human autophagy receptor NBR1 and its interaction with ubiquitin and polyubiquitin.

NBR1 (neighbor of BRCA1 gene 1) is a protein commonly found in ubiquitin-positive inclusions in neurodegenerative diseases. Due to its high architectural similarity to the well studied autophagy receptor protein p62/SQSTM1, NBR1 has been thought to analogously bind to ubiquitin-marked autophagic substrates via its C-terminal ubiquitin-associated (UBA) domain and deliver them to autophagosomes for degradation. Unexpectedly, we find that NBR1 differs from p62 in its UBA structure and accordingly in its interaction with ubiquitin.

Functional assessment of the mutational effects of human IRAK4 and MyD88 genes.

Human interleukin-1 receptor-associated kinase 4 (IRAK4) deficiency and myeloid differentiating factor 88 (MyD88) deficiency syndromes are two primary immune-deficiency disorders with innate immune defects. Although new genetic variations of IRAK4 and MyD88 have recently been deposited in the single nucleotide polymorphism (SNP) database, the clinical significance of these variants has not yet been established. Therefore, it is important to establish methods for assessing the association of each gene variation with human diseases.

Pruning the ALS-associated protein SOD1 for in-cell NMR.

To efficiently deliver isotope-labeled proteins into mammalian cells poses a main challenge for structural and functional analysis by in-cell NMR. In this study we have employed cell-penetrating peptides (CPPs) to deliver the ALS-associated protein superoxide dismutase (SOD1) into HeLa cells. Our results show that, although full-length SOD1 cannot be efficiently internalized, a variant in which the active-site loops IV and VII have been truncated (SOD1(ΔIVΔVII)) yields high cytosolic delivery.

Quantitative comparison of protein dynamics in live cells and in vitro by in-cell (19)F-NMR.

Here we describe how a (19)F-probe incorporated into an endogenous protein by a chemical biology method revealed protein dynamics. By explicit determination of ligand-bound and unbound structures with X-ray crystallography, the quantitative comparison of the protein's dynamics in live cells and in vitro is presented. These results clearly demonstrated the greater conformational fluctuations of the intracellular protein, partially due to macromolecular crowding effects.