Multi-enzyme Machinery for Chitin Degradation in the Chitinolytic Bacterium Chitiniphilus shinanonensis SAY3T.

The chitinolytic bacterium, Chitiniphilus shinanonensis SAY3T was examined to characterize its chitin-degrading enzymes in view of its potential to convert biomass chitin into useful saccharides. A survey of the whole-genome sequence revealed 49 putative genes encoding polypeptides that are thought to be related to chitin degradation. Based on an analysis of the relative quantity of each transcript and an assay for chitin-degrading activity of recombinant proteins, a chitin degradation system driven by 19 chitinolytic enzymes was proposed. These include sixteen endo-type chitinases, two N-acetylglucosaminidases, and one lipopolysaccharide monooxygenase that catalyzes the oxidative cleavage of glycosidic bonds. Among the 16 chitinases, ChiL was characterized by its remarkable transglycosylation activity. Of the two N-acetylglucosaminidases (ChiI and ChiT), ChiI was the major enzyme, corresponding to > 98% of the total cellular activity. Surprisingly, a chiI-disrupted mutant was still able to grow on medium with powdered chitin or GlcNAc dimer. However, its growth rate was slightly lower compared to that of the wild-type SAY3. This multi-enzyme machinery composed of various types of chitinolytic enzymes may support SAY3 to efficiently utilize native chitin as a carbon and energy source and may play a role in developing an enzymatic process to decompose and utilize abundant chitin at the industrial scale.

One-pot synthesis of fibrillar-shaped functional nanomaterial using microbial transglutaminase.

Recently, functional nanowire production using amyloids as a scaffold for protein immobilization has attracted attention. However, protein immobilization on amyloid fibrils often caused protein inactivation. In this study, we investigated protein immobilization using enzymatic peptide ligation to suppress protein inactivation during immobilization. We attempted to immobilize functional molecules such as green fluorescent protein (GFP) and Nanoluc to a transthyretin (TTR) amyloid using microbial transglutaminase (MTG), which links the glutamine side chain to the primary amine. Linkage between amyloid fibrils and functional molecules was achieved through the MTG substrate sequence, and the functional molecules-loaded nanowires were successfully fabricated. We also found that the synthetic process from amyloidization to functional molecules immobilization could be achieved in a single-step procedure.All rights reserved.

Self-Assembling Lectin Nano-Block Oligomers Enhance Binding Avidity to Glycans.

Lectins, carbohydrate-binding proteins, are attractive biomolecules for medical and biotechnological applications. Many lectins have multiple carbohydrate recognition domains (CRDs) and strongly bind to specific glycans through multivalent binding effect. In our previous study, protein nano-building blocks (PN-blocks) were developed to construct self-assembling supramolecular nanostructures by linking two oligomeric proteins. A PN-block, WA20-foldon, constructed by fusing a dimeric four-helix bundle de novo protein WA20 to a trimeric foldon domain of T4 phage fibritin, self-assembled into several types of polyhedral nanoarchitectures in multiples of 6-mer. Another PN-block, the extender PN-block (ePN-block), constructed by tandemly joining two copies of WA20, self-assembled into cyclized and extended chain-type nanostructures. This study developed novel functional protein nano-building blocks (lectin nano-blocks) by fusing WA20 to a dimeric lectin, Agrocybe cylindracea galectin (ACG). The lectin nano-blocks self-assembled into various oligomers in multiples of 2-mer (dimer, tetramer, hexamer, octamer, etc.). The mass fractions of each oligomer were changed by the length of the linkers between WA20 and ACG. The binding avidity of the lectin nano-block oligomers to glycans was significantly increased through multivalent effects compared with that of the original ACG dimer. Lectin nano-blocks with high avidity will be useful for various applications, such as specific cell labeling.

Design of helical linkers for fusion proteins and protein-based nanostructures.

The construction of recombinant fusion/chimeric proteins has been widely used for expression of soluble proteins and protein purification in a variety of fields of protein engineering and biotechnology. Fusion proteins are constructed by the linking of two protein domains with a peptide linker. The selection of a linker sequence is important for the construction of stable and bioactive fusion proteins. Empirically designed linkers are generally classified into two categories according to their structural features: flexible linkers and rigid linkers. Rigid linkers with the α-helix-forming sequences A(EAAAK)nA (n=2-5) were first designed about two decades ago to control the distance between two protein domains and to reduce their interference. Thereafter, the helical linkers have been applied to the construction of many fusion proteins to improve expression and bioactivity. In addition, the design of fusion proteins that self-assemble into supramolecular complexes is useful for nanobiotechnology and synthetic biology. A protein that forms a self-assembling oligomer was fused by a rigid helical linker to another protein that forms another self-assembling oligomer, and the fusion protein symmetrically self-assembled into a designed protein nanoparticle or nanomaterial. Moreover, to construct chain-like polymeric nanostructures, extender protein nanobuilding blocks were designed by tandemly fusing two dimeric de novo proteins with helical or flexible linkers. The linker design of fusion proteins can affect conformation and dynamics of self-assembling nanostructures. The present review and methods focus on useful helical linkers to construct bioactive fusion proteins and protein-based nanostructures.

Design of Hollow Protein Nanoparticles with Modifiable Interior and Exterior Surfaces.

Protein-based nanoparticles hold promise for a broad range of applications. Here, we report the production of a uniform anionic hollow protein nanoparticle, designated TIP60, which spontaneously assembles from a designed fusion protein subunit based on the geometric features of polyhedra. We show that TIP60 tolerates mutation and both its interior and exterior surfaces can be chemically modified. Moreover, TIP60 forms larger structures upon the addition of a cationic protein. Therefore, TIP60 can be used as a modifiable nano-building block for further molecular assembly.

Comparative analysis of plant isochorismate synthases reveals structural mechanisms underlying their distinct biochemical properties.

Isochorismate synthase (ICS) converts chorismate into isochorismate, a precursor of primary and secondary metabolites including salicylic acid (SA). SA plays important roles in responses to stress conditions in plants. Many studies have suggested that the function of plant ICSs is regulated at the transcriptional level. In Arabidopsis thaliana, the expression of AtICS1 is induced by stress conditions in parallel with SA synthesis, and AtICS1 is required for SA synthesis. In contrast, the expression of NtICS is not induced when SA synthesis is activated in tobacco, and it is unlikely to be involved in SA synthesis. Studies on the biochemical properties of plant ICSs are limited, compared with those on transcriptional regulation. We analyzed the biochemical properties of four plant ICSs: AtICS1, NtICS, NbICS from Nicotiana benthamiana, and OsICS from rice. Multiple sequence alignment analysis revealed that their primary structures were well conserved, and predicted key residues for ICS activity were almost completely conserved. However, AtICS1 showed much higher activity than the other ICSs when expressed in Escherichia coli and N. benthamiana leaves. Moreover, the levels of AtICS1 protein expression in N. benthamiana leaves were higher than the other ICSs. Construction and analysis of chimeras between AtICS1 and OsICS revealed that the putative chloroplast transit peptides (TPs) significantly affected the levels of protein accumulation in N. benthamiana leaves. Chimeric and point-mutation analyses revealed that Thr531, Ser537, and Ile550 of AtICS1 are essential for its high activity. These distinct biochemical properties of plant ICSs may suggest different roles in their respective plant species.

Design and construction of self-assembling supramolecular protein complexes using artificial and fusion proteins as nanoscale building blocks.

The central goal of nanobiotechnology is to design and construct novel biomaterials of nanometer sizes. In this short review, we describe recent progress of several approaches for designing and creating artificial self-assembling protein complexes and primarily focus on the following biotechnological strategies for using artificial and fusion proteins as nanoscale building blocks: fusion proteins designed for symmetrical self-assembly; three-dimensional domain-swapped oligomers; self-assembling designed coiled-coil peptide modules; metal-directed self-assembling engineered proteins; computationally designed self-assembling de novo proteins; and self-assembling protein nanobuilding blocks (PN-Blocks) using an intermolecularly folded dimeric de novo protein. These state-of-the-art nanobiotechnologies for designing supramolecular protein complexes will facilitate the development of novel functional nanobiomaterials.

Functional characterization of FABP3, 5 and 7 gene variants identified in schizophrenia and autism spectrum disorder and mouse behavioral studies.

Disturbances of lipid metabolism have been implicated in psychiatric illnesses. We previously reported an association between the gene for fatty acid binding protein 7 (FABP7) and schizophrenia. Furthermore, we identified and reported several rare non-synonymous polymorphisms of the brain-expressed genes FABP3, FABP5 and FABP7 from schizophrenia and autism spectrum disorder (ASD), diseases known to part share genetic architecture. Here, we conducted further studies to better understand the contribution these genes make to the pathogenesis of schizophrenia and ASD. In postmortem brains, we detected altered mRNA expression levels of FABP5 in schizophrenia, and of FABP7 in ASD and altered FABP5 in peripheral lymphocytes. Using a patient cohort, comprehensive mutation screening identified six missense and two frameshift variants from the three FABP genes. The two frameshift proteins, FABP3 E132fs and FABP7 N80fs, formed cellular aggregates and were unstable when expressed in cultured cells. The four missense mutants with predicted possible damaging outcomes showed no changes in intracellular localization. Examining ligand binding properties, FABP7 S86G and FABP7 V126L lost their preference for docosahexaenoic acid to linoleic acid. Finally, mice deficient in Fabp3, Fabp5 and Fabp7 were evaluated in a systematic behavioral test battery. The Fabp3 knockout (KO) mice showed decreased social memory and novelty seeking, and Fabp7 KO mice displayed hyperactive and anxiety-related phenotypes, while Fabp5 KO mice showed no apparent phenotypes. In conclusion, disturbances in brain-expressed FABPs could represent an underlying disease mechanism in a proportion of schizophrenia and ASD sufferers.

Polymorphism screening of brain-expressed FABP7, 5 and 3 genes and association studies in autism and schizophrenia in Japanese subjects.

Fatty acid-binding protein (FABP) gene family encode fatty acid-binding proteins and consist of at least 12 members, of which FABP7, 5 and 3 are expressed in the brain. We previously showed that FABP7 is associated with schizophrenia and bipolar disorder. Recently, genetic overlap between autism and schizophrenia has been reported. Therefore, in this study, we set out to examine the possible roles of brain-expressed FABPs in autism, focusing primarily on potentially functional polymorphisms (that is, missense polymorphisms). First, we resequenced the three genes using 285 autism samples. We identified 13 polymorphisms, of which 7 are novel. Of the novel single-nucleotide polymorphisms (SNPs), two are missense mutations, namely, 376G>C (Val126Leu) in FABP7 and 340G>C (Gly114Arg) in FABP5. Second, we tested for the genetic association of four missense SNPs with autism and schizophrenia, but failed to detect significant results. Finally, as a web-based algorithm predicts that the 8A>G (Asp3Gly; rs17848124) in FABP3 is 'probably damaging', we estimated the possible impact of this SNP, and found that the loss of charge and salt bridge, caused by the Asp3-to-Gly3, may affect stability of the FABP3 protein. Future searches for associated phenotypes with missense SNPs using larger samples are highly warranted.

Conserved protein TTHA1554 from Thermus thermophilus HB8 binds to glutamine synthetase and cystathionine beta-lyase.

TTHA1554 was found as a hypothetical protein composed of 95 amino acids in the genome of the extremely thermophilic bacterium, Thermus thermophilus HB8. Proteins homologous to TTHA1554 are conserved in several bacteria and archaea, although their functions are unknown. To investigate the function of TTHA1554, we identified interacting proteins by using a pull-down assay and mass spectrometry. TTHA1329, which is glutamine synthetase, and TTHA1620, a putative aminotransferase, were identified as TTHA1554 binding proteins. The interactions with TTHA1329 and TTHA1620 were validated using in vitro pull-down assays and surface plasmon resonance biosensor assays with recombinant proteins. Since sequence homology analyses suggested that TTHA1620 was a pyridoxal 5'-phosphate-dependent enzyme, such as an aminotransferase, a cystathionine beta-lyase or a cystalysin, putative substrates were investigated. When cystathionine, cystine and S-methylcysteine were used as substrates, pyruvate was produced by TTHA1620. The data revealed that TTHA1620 has cystathionine beta-lyase enzymatic activity. When TTHA1554 was added to the reaction mixtures, the glutamine synthetase and cystathionine beta-lyase enzymatic activities both increased by approximately two-fold. These results indicated that TTHA1554 is a novel protein (we named it GCBP: glutamine synthetase and cystathionine beta-lyase binding protein) that binds to glutamine synthetase and cystathionine beta-lyase.

Phosphorylation analysis of 90 kDa heat shock protein within the cytosolic arylhydrocarbon receptor complex.

The arylhydrocarbon receptor (AhR) functions as a ligand-activated transcription factor that regulates the transcription of genes encoding xenobiotic metabolizing enzymes and also mediates most of the toxic effects caused by dioxins and polycyclic aromatic hydrocarbons. The cytosolic AhR complex exists as a transcriptionally cryptic complex, consisting of the 90 kDa heat shock protein (HSP90) and the hepatitis B virus X-associated protein 2 (XAP2). The posttranslational modifications, especially phosphorylation, of the cytosolic AhR-HSP90-XAP2 complex are poorly understood, although the phosphorylation of a transcriptionally active heterodimer of AhR and an AhR nuclear translocator is critically involved in AhR function. To reveal the phosphorylation status involved in AhR function, we used mass spectrometry to determine the site-specific phosphorylation of the steady-state cytosolic AhR complex, prepared from Chinese hamster ovary cells stably expressing mouse AhR. We identified phosphorylations of the HSP90 subunits within the AhR complex at Ser225 and Ser254 of HSP90beta and Ser230 of HSP90alpha. By site-directed mutagenesis, these serine residues were substituted with alanine and glutamic acid to elucidate the role of the HSP90beta serine phosphorylations in the AhR function. Immunoprecipitation assays using COS7 transfectants showed that the replacement of Ser225 and Ser254 by Ala, S225/254A, increased the binding affinity for AhR, as compared with the Glu replacement. In a ligand-induced AhR transcription activity assay using Hepa1 transfectants, the S255/254A mutant exhibited more potent transcription activity than the S225/254E mutant, which had activity similar to that of wild-type HSP90beta. These results suggest that the phosphorylations in the charged linker region of the HSP90 molecule modulate the formation of the functional cytosolic AhR complex.