Ribonucleoprotein Y-box-binding protein-1 regulates mitochondrial oxidative phosphorylation (OXPHOS) protein expression after serum stimulation through binding to OXPHOS mRNA

Mitochondria play key roles in essential cellular functions, such as energy production, metabolic pathways and aging. Growth factor-mediated expression of the mitochondrial OXPHOS (oxidative phosphorylation) complex proteins has been proposed to play a fundamental role in metabolic homoeostasis. Although protein translation is affected by general RNA-binding proteins, very little is known about the mechanism involved in mitochondrial OXPHOS protein translation. In the present study, serum stimulation induced nuclear-encoded OXPHOS protein expression, such as NDUFA9 [NADH dehydrogenase (ubiquinone) 1α subcomplex, 9, 39 kDa], NDUFB8 [NADH dehydrogenase (ubiquinone) 1β subcomplex, 8, 19 kDa], SDHB [succinate dehydrogenase complex, subunit B, iron sulfur (Ip)] and UQCRFS1 (ubiquinol-cytochrome c reductase, Rieske iron-sulfur polypeptide 1), and mitochondrial ATP production, in a translation-dependent manner. We also observed that the major ribonucleoprotein YB-1 (Y-box-binding protein-1) preferentially bound to these OXPHOS mRNAs and regulated the recruitment of mRNAs from inactive mRNPs (messenger ribonucleoprotein particles) to active polysomes. YB-1 depletion led to up-regulation of mitochondrial function through induction of OXPHOS protein translation from inactive mRNP release. In contrast, YB-1 overexpression suppressed the translation of these OXPHOS mRNAs through reduced polysome formation, suggesting that YB-1 regulated the translation of mitochondrial OXPHOS mRNAs through mRNA binding. Taken together, our findings suggest that YB-1 is a critical factor for translation that may control OXPHOS activity.     

The RAC binding domain/IRSp53-MIM homology domain of IRSp53 induces RAC-dependent membrane deformation

The concave surface of the crescent-shaped Bin-amphiphysin-Rvs (BAR) domain is postulated to bind to the cell membrane to induce membrane deformation of a specific curvature. The Rac binding (RCB) domain/IRSp53-MIM homology domain (IMD) has a dimeric structure that is similar to the structure of the BAR domain; however, the RCB domain/IMD has a "zeppelin-shaped" dimer. Interestingly, the RCB domain/IMD of IRSp53 possesses Rac binding, membrane binding, and actin filament binding abilities. Here we report that the RCB domain/IMD of IRSp53 induces membrane deformation independent of the actin filaments in a Rac-dependent manner. In contrast to the BAR domain, the RCB domain/IMD did not cause long tubulation of the artificial liposomes; however, the Rac binding domain caused the formation of small buds on the liposomal surface. When expressed in cells, the Rac binding domain induced outward protrusion of the plasma membrane in a direction opposite to that induced by the BAR domain. Mapping of the amino acids responsible for membrane deformation suggests that the convex surface of the Rac binding domain binds to the membrane in a Rac-dependent manner, which may explain the mechanism of the membrane deformation induced by the RCB domain/IMD.     

Solution structure of the kinase-associated domain 1 of mouse microtubule-associated protein/microtubule affinity-regulating kinase 3

Microtubule-associated protein/microtubule affinity-regulating kinases (MARKs)/PAR-1 are common regulators of cell polarity that are conserved from nematode to human. All of these kinases have a highly conserved C-terminal domain, which is termed the kinase-associated domain 1 (KA1), although its function is unknown. In this study, we determined the solution structure of the KA1 domain of mouse MARK3 by NMR spectroscopy. We found that approximately 50 additional residues preceding the previously defined KA1 domain are required for its proper folding. The newly defined KA1 domain adopts a compact alpha+beta structure with a betaalphabetabetabetabetaalpha topology. We also found a characteristic hydrophobic, concave surface surrounded by positively charged residues. This concave surface includes the highly conserved Glu-Leu-Lys-Leu motif at the C terminus, indicating that it is important for the function of the KA1 domain.     

Production of functional bacteriorhodopsin by an Escherichia coli cell‐free protein synthesis system supplemented with steroid detergent and lipid

Cell‐free expression has become a highly promising tool for the efficient production of membrane proteins. In this study, we used a dialysis‐based Escherichia coli cell‐free system for the production of a membrane protein actively integrated into liposomes. The membrane protein was the light‐driven proton pump bacteriorhodopsin, consisting of seven transmembrane α‐helices. The cell‐free expression system in the dialysis mode was supplemented with a combination of a detergent and a natural lipid, phosphatidylcholine from egg yolk, in only the reaction mixture. By examining a variety of detergents, we found that the combination of a steroid detergent (digitonin, cholate, or CHAPS) and egg phosphatidylcholine yielded a large amount (0.3–0.7 mg/mL reaction mixture) of the fully functional bacteriorhodopsin. We also analyzed the process of functional expression in our system. The synthesized polypeptide was well protected from aggregation by the detergent‐lipid mixed micelles and/or lipid disks, and was integrated into liposomes upon detergent removal by dialysis. This approach might be useful for the high yield production of functional membrane proteins.     

Clock genes regulate neurogenic transcription factors,including NeuroD1, and the neuronal differentiation of adult neural stem/progenitor cells

The circadian clock system plays multiple roles in our bodies, and clock genes are expressed in various brain regions, including the lateral subventricular zone (SVZ) where neural stem/progenitor cells (NSPCs) persist and postnatal neurogenesis continues. However, the functions of clock genes in adult NSPCs are not well understood. Here, we first investigated the expression patterns of Clock and Bmal1 in the SVZ by immunohistochemistry and then verified how the expression levels of 17 clock and clock-related genes changed during differentiation of cultured adult NSPCs using quantitative RT-PCR. Finally, we used RNAi to observe the effects of Clock and Bmal1 on neuronal differentiation. Our results revealed that Clock and Bmal1 were expressed in the SVZ and double-stained with the neural progenitor marker Nestin and neural stem marker GFAP. In cultured adult NSPCs, the clock genes changed their expression patterns during differentiation, and interestingly, Bmal1 started endogenous oscillation. Moreover, gene silencing of Clock or Bmal1 by RNAi decreased the percentages of neuronal marker Map2-positive cells and expression levels of NeuroD1 mRNA. These findings suggest that clock genes are involved in the neuronal differentiation of adult NSPCs and may extend our understanding of various neurological/psychological disorders linked to adult neurogenesis and circadian rhythm.     

NMR and X-ray structures of the putative sterol carrier protein 2 from <Emphasis Type="Italic">Thermus thermophilus</Emphasis> HB8 show conformational changes

Sterol carrier protein 2 (SCP-2), also known as nonspecific lipid transfer protein, is a ubiquitous intracellular ~13 kDa protein found in mammals, insects, plants, archaea, and bacteria. Vertebrate SCP-2 has been implicated in a wide range of lipid-related functions in vitro, although its actual physiological role is still unknown. Tunnels in the protein serve as fatty acid binding vehicles. Here we report the first putative SCP-2 structure from a bacterium: specifically, the NMR and X-ray structures of the TTHA0401 protein (also designated as TT1886) from the extremely thermophilic bacterium Thermus thermophilus. The NMR structure and the two chain structures (chain A and chain B) of the asymmetric crystallographic unit (space group (P2₁2₁2₁)) revealed an internal cavity. However, this cavity is open to the outside, forming a tunnel, in only one of those structures (chain A, X-ray). The location of this tunnel is different from the one found in other SCP-2 proteins, and inaccessible cavities have not been seen before in SCP structures. We present evidence that at physiological concentrations, TTHA0401 likely exists as a monomer in equilibrium between open and closed conformations. This equilibrium is influenced by temperature-dependent dynamics, and is likely to be very different at the high temperatures preferred by this hyperthermophilic bacterium. Alternatively, another protein binding to TTHA0401 may induce a conformational change, which would constitute an intriguing metabolic regulation method in bacteria.     

Surfactant sodium lauryl sulfate enhances skin vaccination: molecular characterization via a novel technique using ultrafiltration capillaries and mass spectrometric proteomics

The skin is a highly accessible organ and thus provides an attractive immune environment for cost-effective, simple, and needle-free delivery of vaccines and immunomodulators. In this study, we pretreated mouse skin with an anionic surfactant, sodium lauryl sulfate (SLS), for a short period of time (10 min) followed by epicutaneous vaccination with hen egg lysozyme antigen. We demonstrated for the first time that pretreatment of skin with surfactant SLS significantly enhances the production of antibody to hen egg lysozyme. Short term pretreatment with SLS disorganized the stratum corneum, extracted partial lamellar lipids, induced the maturation of Langerhans cells, and did not result in epidermis thickening. To reveal the mechanism underlying these changes, particularly at the molecular level, we used a novel proteomic technique using ultrafiltration capillaries and mass spectrometry to identify in vivo proteins/peptides secreted in the SLS-pretreated skin. Two secretory proteins, named as calcium-binding protein S100A9 and thymosin beta4, were identified by this novel technique. These two proteins thus may provide new insight into the enhancing effect of surfactants on skin vaccination.