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Preview: Journal of Biochemistry - current issue

The Journal of Biochemistry Current Issue





Published: Thu, 20 Jul 2017 00:00:00 GMT

Last Build Date: Tue, 29 Aug 2017 09:48:23 GMT

 



Role of the unfolded protein response in the development of central nervous system

2017-07-20

Abstract
The unfolded protein response (UPR) is an intracellular homeostatic signalling pathway that is induced by accumulated misfolded/unfolded proteins in the endoplasmic reticulum (ER). The UPR is closely associated with the development of disease in several tissues, including the central nervous system (CNS), in response to ER stress. More recently, the unique features and importance of the UPR have been revealed in neural stem cells (NSCs) and differentiated CNS cells [neurons and glial cells (astrocytes and oligodendrocytes)]. Although several UPR signalling pathways dynamically change in each CNS cell during brain development, the role of UPR signalling in CNS cells (especially NSCs and glial cells) under pathological or physiological conditions is poorly understood. Here, we discuss and summarize the recent progress in understanding how the UPR regulates the proliferation, differentiation, maturation and viability of CNS cells.



ERK signalling as a regulator of cell motility

2017-07-11

Abstract
Cell motility is regulated by multiple processes, including cell protrusion, cell retraction, cell–matrix adhesion, polarized exocytosis and polarized vesicle trafficking, each of which is spatiotemporally controlled by various intracellular signalling pathways. Dysregulation of cell motility leads to pathological conditions, such as tumour invasion and metastasis. Accumulating evidence has revealed that extracellular signal-regulated kinase (ERK) signalling is one of the critical regulators of cell motility, although it is classically known as an important regulator of cell proliferation, differentiation and survival through regulation of gene expression. ERK and its downstream kinase, p90 ribosomal S6 kinase (RSK), dynamically regulate cell motility mainly through direct phosphorylation of various molecules that are not necessarily involved in the regulation of gene transcription and translation. In this review, we summarize how ERK signalling regulates cell motility by focusing on the components of the cell motility machinery that are directly regulated by ERK or RSK.



Use of SpyTag/SpyCatcher to construct bispecific antibodies that target two epitopes of a single antigen

2017-06-16

Abstract
Bispecific antibody targeting of two different antigens is promising, but when fragment-based antibodies are used, homogeneous production is difficult. To overcome this difficulty, we developed a method using the SpyTag/SpyCatcher system in which a covalent bond is formed between the two polypeptides. Using this method, we constructed a bispecific antibody that simultaneously interacted with two different epitopes of roundabout homologue 1 (ROBO1), a membrane protein associated with cancer progression. A bispecific tetravalent antibody with an additional functional moiety was also constructed by using a dimeric biotin-binding protein. An interaction analysis of ROBO1-expressing cells and the recombinant antigen demonstrated the improved binding ability of the bispecific antibodies through spontaneous binding of the two antibody fragments to their respective epitopes. In addition, multivalency delayed dissociation, which is advantageous in therapy and diagnosis.



Effects of various spacers between biotin and the phospholipid headgroup on immobilization and sedimentation of biotinylated phospholipid-containing liposomes facilitated by avidin–biotin interactions

2017-04-24

Abstract
Immobilization and sedimentation of liposomes (lipid vesicles) are used in liposome-protein binding assays, facilitated by avidin/streptavidin/NeutrAvidin and biotinylated phospholipid-containing liposomes. Here, we examined the effects of three spacers [six-carbon (X), polyethylene glycol (PEG) 180 (molecular weight 180) and PEG2000 (molecular weight 2,000)] between biotin and the phospholipid headgroup on the immobilization and sedimentation of small unilamellar liposomes/vesicles (SUVs). PEG180 and PEG2000 showed more efficient immobilization of biotinylated SUVs on NeutrAvidin-coated plates than X, but X and PEG180 showed more efficient sedimentation of biotinylated SUVs upon NeutrAvidin addition than PEG2000. Thus, the most appropriate spacers differed between immobilization and sedimentation. A spacer for biotinylated SUVs must be selected according to the particular liposome-protein binding assays examined.



Effects of neutral salts and pH on the activity and stability of human RNase H2

2017-04-11

Abstract
Ribonuclease H (RNase H) specifically degrades the RNA of RNA/DNA hybrid. Recent study has shown that a single ribonucleotide is embedded in DNA double strand at every few thousand base pairs in human genome, and human RNase H2 is involved in its removal. Here, we examined the effects of neutral salts and pH on the activity and stability of human RNase H2. NaCl, KCl, RbCl and NaBr increased the activity to 170–390% at 10–60 mM, while LiCl, LiBr and CsCl inhibited it, suggesting that species of cation, but not anion, is responsible for the effect on activity. NaCl and KCl increased the stability by decreasing the first-order rate constant of the inactivation to 50–60% at 60–80 mM. The activity at 25–35 °C exhibited a narrow bell-shaped pH-dependence with the acidic and alkaline pKe (pKe1 and pKe2) values of 7.3 − 7.6 and 8.1 − 8.8, respectively. Enthalpy changes (ΔH°) of deprotonation were 5 ± 21 kJ mol−1 for pKe1 and 68 ± 25 kJ mol−1 for pKe2. These results suggest that the ionizable groups responsible for pKe1 may be two out of Asp34, Glu35 and Asp141 of DEDD motif, and that for pKe2 may be Lys69 of DSK motif.



Order of stability for proteolysis sites of a bacterial collagen-like protein

2017-03-23

Abstract
When compared with collagens isolated from animal sources, collagens and collagen-like (CL) proteins from non-animal sources are non-immunogenic and thus promising as biomedical materials. Recently, a CL protein, V-CL, was identified from a bacterial source, Streptococcus pyogenes. In this study, an acid-precipitation method was used to isolate V-CL in one purification step. Circular dichroism spectroscopy was used to examine the triple-helix structure of V-CL. The trypsin proteolysis events of V-CL were characterized using gel electrophoresis and mass spectrometry. The proteolysis order of the proteolysis sites of V-CL was investigated and compared with the predicted stability profile. The experimentally determined proteolysis order agreed with the predicted stability profile, suggesting that the proteolysis order of these proteolysis sites was determined by their structural stability. This work is among the pioneer studies on establishing a correlation between the order of proteolysis and the predicted protein stability profile of collagens and CL proteins. Since the digestion of collagen is frequently linked to disease states, this study can potentially shed light on the biodegradation pathway of collagens and related disease models. Moreover, as collagens have been used in drug delivery, this work can provide the molecular basis for rational design of CL proteins with varied biostability.



Assessment of direct interaction between CD36 and an oxidized glycerophospholipid species

2017-03-14

Abstract
Cluster of differentiation 36 (CD36) is a transmembrane protein that recognizes multiple diverse ligands. It is believed that (i) oxidized glycerophosphatidylcholine species having a terminal γ-hydroxyl(or oxo)-α,β-unsaturated carbonyl on the sn-2 acyl group (oxGPCCD36), which can occur on the surface of lipoprotein particles, serve as high-affinity ligands for CD36, and (ii) the amino acid 150–168 of CD36 (CD36150–168) is responsible for recognizing oxGPCCD36. However, it remains uncertain whether CD36150–168 directly interacts with oxGPCCD36 alone. In this study, we addressed this issue by investigating and comparing the banding pattern by non-denaturing polyacrylamide gel electrophoresis of a glutathione S-transferase (GST) fusion protein containing CD36150–168 (GST-CD36150–168), in the presence and absence of an oxGPCCD36 species, 1-(palmitoyl)-2-(5-keto-6-octenedioyl)phosphatidylcholine (KOdiA-PC). It was shown that GST-CD36150–168 pre-incubated with KOdiA-PC produced bands at upper positions than did the fusion protein alone. Further analyses revealed that the bands produced by the loading of GST-CD36150–168/KOdiA-PC mixture represent complexes consisting of the fusion protein and lipid. To our knowledge, this is the first evidence for direct interaction between CD36150–168 and oxGPCCD36 alone. It is also notable that the electrophoresis-based technique provides a convenient means to evaluate protein–lipid interactions.



A polygalacturonase localized in the Golgi apparatus in Pisum sativum

2017-02-23

Abstract
Pectin is a plant cell wall constituent that is mainly composed of polygalacturonic acid (PGA), a linear α1,4-d-galacturonic acid (GalUA) backbone. Polygalacturonase (PG) hydrolyzes the α1,4-linkages in PGA. Nearly all plant PGs identified thus far are secreted as soluble proteins. Here we describe the microsomal PG activity in pea (Pisum sativum) epicotyls and present biochemical evidence that it was localized to the Golgi apparatus, where pectins are biosynthesized. The microsomal PG was purified, and it was enzymatically characterized. The purified enzyme showed maximum activity towards pyridylaminated oligogalacturonic acids with six degrees of polymerization (PA-GalUA6), with a Km value of 11 μM for PA-GalUA6. The substrate preference of the enzyme was complementary to that of PGA synthase. The main PG activity in microsomes was detected in the Golgi fraction by sucrose density gradient ultracentrifugation. The activity of the microsomal PG was lower in rapidly growing epicotyls, in contrast to the high expression of PGA synthase. The role of this PG in the regulation of pectin biosynthesis or plant growth is discussed.



Purification and enzymatic characterization of Gallus gallus BLM helicase

2017-02-21

Abstract
Mutations in human BLM helicase give rise to the autosomal recessive Bloom syndrome, which shows high predisposition to types of malignant tumours. Though lots of biochemical and structural investigations have shed lights on the helicase core, structural investigations of the whole BLM protein are still limited due to its low stability and production. Here by comparing with the expression systems and functions of other BLM homologues, we developed the heterologous high-level expression and high-yield purification systems for Gallus gallus BLM (gBLM) in Escherichia coli. Subsequent DNA binding and unwinding determinations demonstrated that gBLM was a vigorous atypical DNA structure specific helicase, which not only showed high preference for the 3′-tailed DNA structures but also could efficiently unwind bubble DNA structures with blunt-ends, indicating its biological roles in processing DNA metabolism intermediates. Further comparative analysis between gBLM and gBLM Core revealed that the long N-terminal domain facilitated the binding affinity of forked and bubble DNA structures and it was also required for the DNA unwinding activities of gBLM. Thus, we present the first enzymatic characterization of gBLM and its N-terminal domain, providing a new model for probing the mechanism and structure of human BLM.



Crystal structure of metagenomic β-xylosidase/ α- l -arabinofuranosidase activated by calcium

2017-02-16

Abstract
The crystal structure of metagenomic β-xylosidase/α-l-arabinofuranosidase CoXyl43, activated by calcium ions, was determined in its apo and complexed forms with xylotriose or l-arabinose in the presence and absence of calcium. The presence of calcium ions dramatically increases the kcat of CoXyl43 for p-nitrophenyl β-d-xylopyranoside and reduces the Michaelis constant for p-nitrophenyl α-l-arabinofuranoside. CoXyl43 consists of a single catalytic domain comprised of a five-bladed β-propeller. In the presence of calcium, a single calcium ion was observed at the centre of this catalytic domain, behind the catalytic pocket. In the absence of calcium, the calcium ion was replaced with one sodium ion and one water molecule, and the positions of these cations were shifted by 1.3 Å. The histidine-319 side chain, which coordinates to the 2-hydroxyl oxygen atom of the bound xylose molecule in the catalytic pocket, also coordinates to the calcium ion, but not to the sodium ion. The calcium-dependent increase in activity appears to be caused by the structural change in the catalytic pocket induced by the tightly bound calcium ion and coordinating water molecules, and by the protonation state of glutamic acid-268, the catalytic acid of the enzyme. Our findings further elucidate the complex relationship between metal ions and glycosidases.