Flexibility of GroES Mobile Loop Is Required for Efficient Chaperonin Function

Chaperonin GroEL and its partner GroES assist the folding of nascent and stress-damaged proteins in an ATP-dependent manner. Free GroES has a flexible "mobile loop" and binds to GroEL through the residues at the tip of the loop, capping the central cavity of GroEL to provide the substrate polypeptide a cage for secure in-cage folding. Here, we show that restriction of the flexibility of the loop by a disulfide cross-linking between cysteines within the loop results in the inefficient formation of a stable GroEL-polypeptide-GroES ternary complex and inefficient folding. Then, we generated substrate proteins with enhanced binding affinity to GroEL by fusion of one or two SBP (strongly binding peptide for GroEL) sequences and examined the effect of disulfide cross-linking on the assisted folding. The results indicate that the higher the binding affinity of the substrate polypeptide to GroEL, the greater the contribution of the mobile loop flexibility to efficient in-cage folding. It is likely that the flexibility helps GroES capture GroEL's binding sites that are already occupied by the substrate polypeptide with various binding modes.     

The Monomer-Seed Interaction Mechanism in the Formation of the β2-Microglobulin Amyloid Fibril Clarified by Solution NMR Techniques

Amyloid fibrils are proteinous aggregates associated with various diseases, including Alzheimer's disease, type II diabetes, and dialysis-related amyloidosis. It is generally thought that, during the progression of these diseases, a precursor peptide or protein assumes a partially denatured structure, which interacts with the fibril seed to change into the final amyloid form. β2-Microglobulin (β2m), associated with dialysis-related amyloidosis, is known to form amyloid fibrils at low pH via a partially structured state. However, the molecular mechanism by which the conformation of β2m changes from the precursor to the final fibril structure is poorly understood. We performed various NMR experiments to characterize acid-denatured β2m. The analysis of the transverse relaxation rates revealed that acid-denatured β2m undergoes a structural exchange with an extensively unfolded form. The results of transferred cross-saturation experiments indicated that residues with a residual structure in the acid-denatured state are associated with the interaction with the fibril seed. Our experimental data suggest the partially structured state to be "activated" to become extensively unfolded, in which state the hydrophobic residues are exposed and associate with the seed. Our results provide general information about the extension of amyloid fibrils.     

Three-dimensional structure of nylon hydrolase and mechanism of nylon-6 hydrolysis

We performed x-ray crystallographic analyses of the 6-aminohexanoate oligomer hydrolase (NylC) from Agromyces sp. at 2.0 Å-resolution. This enzyme is a member of the N-terminal nucleophile hydrolase superfamily that is responsible for the degradation of the nylon-6 industry byproduct. We observed four identical heterodimers (27 kDa + 9 kDa), which resulted from the autoprocessing of the precursor protein (36 kDa) and which constitute the doughnut-shaped quaternary structure. The catalytic residue of NylC was identified as the N-terminal Thr-267 of the 9-kDa subunit. Furthermore, each heterodimer is folded into a single domain, generating a stacked αββα core structure. Amino acid mutations at subunit interfaces of the tetramer were observed to drastically alter the thermostability of the protein. In particular, four mutations (D122G/H130Y/D36A/E263Q) of wild-type NylC from Arthrobacter sp. (plasmid pOAD2-encoding enzyme), with a heat denaturation temperature of T_m = 52 °C, enhanced the protein thermostability by 36 °C (T_m = 88 °C), whereas a single mutation (G111S or L137A) decreased the stability by ∼10 °C. We examined the enzymatic hydrolysis of nylon-6 by the thermostable NylC mutant. Argon cluster secondary ion mass spectrometry analyses of the reaction products revealed that the major peak of nylon-6 (m/z 10,000–25,000) shifted to a smaller range, producing a new peak corresponding to m/z 1500–3000 after the enzyme treatment at 60 °C. In addition, smaller fragments in the soluble fraction were successively hydrolyzed to dimers and monomers. Based on these data, we propose that NylC should be designated as nylon hydrolase (or nylonase). Three potential uses of NylC for industrial and environmental applications are also discussed.     

Axonal gradient of arachidonic acid-containing phosphatidylcholine and its dependence on actin dynamics

Phosphatidylcholine (PC) is the most abundant component of lipid bilayers and exists in various molecular forms, through combinations of two acylated fatty acids. Arachidonic acid (AA)-containing PC (AA-PC) can be a source of AA, which is a crucial mediator of synaptic transmission and intracellular signaling. However, the distribution of AA-PC within neurons has not been indicated. In the present study, we used imaging mass spectrometry to characterize the distribution of PC species in cultured neurons of superior cervical ganglia. Intriguingly, PC species exhibited a unique distribution that was dependent on the acyl chains at the sn-2 position. In particular, we found that AA-PC is enriched within the axon and is distributed across a proximal-to-distal gradient. Inhibitors of actin dynamics (cytochalasin D and phallacidin) disrupted this gradient. This is the first report of the gradual distribution of AA-PC along the axon and its association with actin dynamics.     

Structure and properties of the recombinant NADH-cytochrome b5 reductase of Physarum polycephalum

A cDNA for NADH-cytochrome b(5) reductase of Physarum polycephalum was cloned from a cDNA library, and the nucleotide sequence of the cDNA was determined (accession no. AB259870). The DNA of 943 base pairs contains 5'- and 3'-noncoding sequences, including a polyadenylation sequence, and a coding sequence of 843 base pairs. The amino acid sequence (281 residues) deduced from the nucleotide sequence was 25 residues shorter than those of vertebrate enzymes. Nevertheless, the recombinant Physarum enzyme showed enzyme activity comparable to that of the human enzyme. The recombinant Physarum enzyme showed a pH optimum of around 6.0, and apparent K(m) values of 2 microM and 14 microM for NADH and cytochrome b(5) respectively. The purified recombinant enzyme showed a typical FAD-derived absorption peak of cytochrome b(5) reductase at around 460 nm, with a shoulder at 480 nm. These results suggest that the Physarum enzyme plays an important role in the organism.     

A pilot crossover study: effects of an intervention using an activity monitor with computerized game functions on physical activity and body composition

Background

Wearing an activity monitor as a motivational tool and incorporating a behavior-based reward system or a computerized game element might have a synergistic effect on an increase in daily physical activity, thereby inducing body fat reduction. This pilot crossover study aimed to examine the effects of a short-term lifestyle intervention using an activity monitor with computerized game functions on physical activity and body composition.

Methods

Twenty healthy volunteers (31 ± 3 years) participated in a 12-week crossover study. The participants were randomly assigned to either Group A (a 6-week game intervention followed by a 6-week normal intervention) or Group B (a 6-week normal intervention followed by a 6-week game intervention). The participants wore both a normal activity monitor (Lifecorder EX) and an activity monitor with computerized game functions (Yuuhokei) during the game intervention, whereas they only wore a normal activity monitor during the normal intervention. Before, during, and after the intervention, body composition was assessed.

Results

Significantly more daily steps were recorded for the game intervention than for the normal intervention (10,520 ± 562 versus 8,711 ± 523 steps/day, P < 0.01). The participants performed significantly more physical activity at an intensity of ≥ 3 metabolic equivalents (METs) in the game intervention than in the normal intervention (3.1 ± 0.2 versus 2.4 ± 0.2 METs · hour/day, P < 0.01). Although body mass and fat were significantly reduced in both periods (P < 0.01), the difference in body fat reduction was significantly greater in the game intervention than in the normal intervention (P < 0.05).

Conclusions

A short-term intervention using an activity monitor with computerized game functions increases physical activity and reduces body fat more effectively than an intervention using a standard activity monitor.     

TTLL10 can perform tubulin glycylation when co-expressed with TTLL8

Tubulin can undergo unusual post-translational modifications, glycylation and glutamylation. We previously failed to find glycylase (glycine ligase) for tubulin while identifying TTLL10 as a polyglycylase for nucleosome assembly protein 1. We here examine whether TTLL10 performs tubulin glycylation. We used a polyclonal antibody (R-polygly) raised against a poly(glycine) chain, which does not recognize monoglycylated protein. R-polygly strongly reacted with mouse tracheal cilia and axonemal tubulins. R-polygly detected many proteins in cell lysates co-expressing TTLL10 with TTLL8. Two-dimensional electrophoresis revealed that the R-polygly-reactive proteins included α- and β-tubulin. R-polygly labeling signals overlapped with microtubules. These results indicate that TTLL10 can strongly glycylate tubulin in a TTLL8-dependent manner. Furthermore, these two TTLL proteins can glycylate unidentified 170-, 110-, 75-, 40-, 35-, and 30-kDa acidic proteins.     

<Emphasis Type="Italic">Caenorhabditis elegans</Emphasis> chromosome arms are anchored to the nuclear membrane via discontinuous association with LEM-2

Background  

Although Caenorhabditis elegans was the first multicellular organism with a completely sequenced genome, how this genome is arranged within the nucleus is not known.     

Direct observation of minimum-sized amyloid fibrils using solution NMR spectroscopy

It is challenging to investigate the structure and dynamics of amyloid fibrils at the residue and atomic resolution because of their high molecular weight and heterogeneous properties. Here, we used solution nuclear magnetic resonance (NMR) spectroscopy to characterize the conformation and flexibility of amyloid fibrils of β2-microglobulin (β2m), for which direct observation of solution NMR could not be made. Ultrasonication led to fragmentation producing a solution of minimum-sized fibrils with a molecular weight of around 6 MDa. In 1H-15N heteronuclear single-quantum correlation measurements, five signals, derived from N-terminal residues (i.e., Ile1, Gln2, Arg3, Thr4, and Lys6), were newly detected. Signal strength decreased with the distance from the N-terminal end. Capping experiments with the unlabeled β2m monomer indicated that the signals originated from molecules located inside the fibrils. Ultrasonication makes the residues with moderate flexibility observable by reducing size of the fibrils. Thus, solution NMR measurements of ultrasonicated fibrils will be promising for studying the structure and dynamics of fibrils.