Small heat shock protein (sHsp) is a molecular chaperone with a conserved alpha-crystallin domain that can prevent protein aggregation. It has been shown that sHsps exist as oligomers (12-40 mer) and their dissociation into small dimers or oligomers is functionally important. Since several sHsps are upregulated and co-localized with amyloid-β (Aβ) in senile plaques of patients with Alzheimer's disease (AD), sHsps are thought to be involved in AD. Previous studies have also shown that sHsp can prevent Aβ aggregation in vitro. However, it remains unclear how the quaternary structure of sHsp influences Aβ aggregation. In this study, we report for the first time the effect of the quaternary structure of sHsp on Aβ aggregation using sHsp from the fission yeast Schizosaccharomyces pombe (SpHsp16.0) showing a clear temperature-dependent structural transition between an oligomer (30 °C) and dimer (50 °C) state. Aβ aggregation was inhibited by the oligomeric form of SpHsp16.0. In contrast, amyloid fibrils were formed in the presence of dimeric SpHsp16.0. Interestingly, these amyloid fibrils consisted of both Aβ and SpHsp16.0 and showed a low ThT intensity and low cytotoxicity due to their low binding affinity to the cell surface. These results suggest the formation of novel fibrillar Aβ amyloid with different characteristics from that of the authentic Aβ amyloid fibrils formed in the absence of sHsp. Our results also suggest the potential protective role of sHsp in AD under stress conditions.

Reductive dehalogenase (RDase) consists of two parts, RdhA and RdhB. RdhA is the catalytic subunit, harboring a cobalamin cofactor and two Fe-S clusters. RdhA is anchored to the cytoplasmic membrane via the membrane anchoring subunit, RdhB. There are many genes encoding RDases in the genome of organohalide-respiring bacteria, including Dehalococcoides spp. However, most genes have not been functionally characterized. Biochemical studies on RDases have been hampered by difficulties encountered in their expression and purification. In this study, we have expressed, purified and characterized RdhA of RDase for tetrachloroethene (PceA) from Geobacter sp. PceA was expressed as a fusion protein with a trigger factor tag in Escherichia coli. PceA was purified and denatured in aerobic condition. Subsequently, this protein was refolded in the presence of FeCl3, Na2S and cobalamin in anaerobic condition. The reconstituted PceA exhibited dechlorination ability for tetrachloroethene. UV-Vis spectroscopy has shown that it contains cobalamin and Fe-S clusters. Since this method requires anaerobic manipulation only in the reconstituting process and has a relatively high yield, it will enable further biochemical studies of RDases.

Each of the olfactory sensory neurons (OSNs) chooses to express a single G protein-coupled olfactory receptor (OR) from a pool of hundreds. Here, we show the receptor transporting protein (RTP) family members play a dual role in both normal OR trafficking and determining OR gene choice probabilities. Rtp1 and Rtp2 double knockout mice (RTP1,2DKO) show OR trafficking defects and decreased OSN activation. Surprisingly, we discovered a small subset of the ORs are expressed in larger numbers of OSNs despite the presence of fewer total OSNs in RTP1,2DKO. Unlike typical ORs, some overrepresented ORs show robust cell surface expression in heterologous cells without the co-expression of RTPs. We present a model in which developing OSNs exhibit unstable OR expression until they choose to express an OR that exits the ER or undergo cell death. Our study sheds light on the new link between OR protein trafficking and OR transcriptional regulation.

Previously, we demonstrated the ATP-dependent dynamics of a group II chaperonin at the single-molecule level by diffracted X-ray tracking (DXT). The disadvantage of DXT is that it requires a strong X-ray source and also perfect gold nano-crystals. To resolve this problem, we developed diffracted electron tracking (DET). Electron beams have scattering cross-sections that are approximately 1000 times larger than those of X-rays. Thus, DET enables us to perform super-accurate measurements of the time-resolved 3D motion of proteins labeled with commercially available gold nanorods using a scanning electron microscope. In this study, we compared DXT and DET using the group II chaperonin from Methanococcus maripaludis (MmCpn) as a model protein. In DET, the samples are prepared in an environmental cell (EC). To reduce the electron beam-induced protein damage, we immobilized MmCpn on the bottom of the EC to expose gold nanorods close to the carbon thin film. The sample setup worked well, and the motions of gold nanorods were clearly traced. Compared with the results of DXT, the mobility in DET was significantly higher, which is probably due to the difference in the method for immobilization. In DET, MmCpn was immobilized on a film of triacetyl cellulose. Whereas proteins are directly attached on the surface of solid support in DXT. Therefore, MmCpn could move relatively freely in DET. DET will be a state-of-the-art technology for analyzing protein dynamics.

Receptor-transporting protein 1S (RTP1S) is an accessory protein that mediates the transport of mammalian odorant receptors (ORs) into the plasma membrane. Although most ORs fail to localize to the cell surface when expressed alone in nonolfactory cells, functional expression of ORs is achieved with the coexpression of RTP1S. However, the mechanism for RTP1S-mediated OR trafficking remains unclear. In this study, we attempted to reveal the mode of action and critical residues of RTP1S in OR trafficking. Experiments using N-terminal truncation and Ala substitution mutants of RTP1S demonstrated that four N-terminal amino acids have essential roles in OR trafficking. Additionally, using recombinant proteins and split luciferase assays in mammalian cells, we provided evidence for the dimer formation of RTP1S. Furthermore, we determined that the 2nd Cys residue is required for the efficient dimerization of RTP1S. Altogether, these findings provide insights into the mechanism for plasma membrane transport of ORs by RTP1S.

The eukaryotic group II chaperonin, the chaperonin-containing t-complex polypeptide 1 (CCT), plays an important role in cytosolic proteostasis. It has been estimated that as much as 10% of cytosolic proteins interact with CCT during their folding process. CCT is composed of 8 different paralogous subunits. Due to its complicated structure, molecular and biochemical investigations of CCT have been difficult. In this study, we constructed an expression system for CCT from a thermophilic fungus, Chaetomium thermophilum (CtCCT), by using E. coli as a host. As expected, we obtained recombinant CtCCT with a relatively high yield, and it exhibited fairly high thermal stability. We showed the advantages of the overproduction system by characterizing CtCCT variants containing ATPase-deficient subunits. For diffracted X-ray tracking experiment, we removed all surface exposed cysteine residues, and added cysteine residues at the tip of helical protrusions of selected two subunits. Gold nanocrystals were attached onto CtCCTs via gold-thiol bonds and applied for the analysis by diffracted X-ray tracking. Irrespective of the locations of cysteines, it was shown that ATP binding induces tilting motion followed by rotational motion in the CtCCT molecule, like the archaeal group II chaperonins. When gold nanocrystals were attached onto two subunits in the high ATPase activity hemisphere, the CtCCT complex exhibited a fairly rapid response to the motion. In contrast, the response of CtCCT, which had gold nanocrystals attached to the low-activity hemisphere, was slow. These results clearly support the possibility that ATP-dependent conformational change starts with the high-affinity hemisphere and progresses to the low-affinity hemisphere.

Anti-inflammatory drugs inhibit inflammation, particularly those classified as nonsteroidal anti-inflammatory drugs (NSAIDs). Several studies have reported that propolis has both anti-ulcerogenic and anti-inflammatory effects. In this study, we investigated the bioactive compound and in vivo anti-inflammatory properties of both smooth and rough propolis from Tetragronula sp. To further identify anti-inflammatory markers in propolis, LC-MS/MS was used, and results were analyzed by Mass Lynx 4.1. Rough and smooth propolis of Tetragonula sp. were microcapsulated with maltodextrin and arabic gum. Propolis microcapsules at dose 25-200 mg/kg were applied for carrageenan-induced rat's paw-inflammation model. Data were analyzed by one-way ANOVA and Kruskal-Wallis statistical tests. LC-MS/MS experiments identified seven anti-inflammatory compounds, including [6]-dehydrogingerdione, alpha-tocopherol succinate, adhyperforin, 6-epiangustifolin, deoxypodophyllotoxin, kurarinone, and xanthoxyletin. Our results indicated that smooth propolis at 50 mg/kg inhibited inflammation to the greatest extent, followed by rough propolis at a dose of 25 mg/kg. SPM and RPM with the dose of 25 mg/kg had inflammatory inhibition value of 62.24% and 58.12%, respectively, which is comparable with the value 70.26% of sodium diclofenac with the dose of 135 mg/kg. This study suggests that propolis has the potential candidate to develop as a non-steroid anti-inflammatory drug.

We report a thermodynamic and structural analysis of six extensively simplified bovine pancreatic trypsin inhibitor (BPTI) variants containing 19-24 alanines out of 58 residues. Differential scanning calorimetry indicated a two-state thermal unfolding, typical of a native protein with densely packed interior. Surprisingly, increasing the number of alanines induced enthalpy stabilization, which was however over-compensated by entropy destabilization. X-ray crystallography indicated that the alanine substitutions caused the recruitment of novel water molecules facilitating the formation of protein-water hydrogen bonds and improving the hydration shells around the alanine's methyl groups, both of which presumably contributed to enthalpy stabilization. There was a strong correlation between the number of water molecules and the thermodynamic parameters. Overall, our results demonstrate that, in contrast to our initial expectation, a protein sequence in which over 40% of the residues are alanines can retain a densely packed structure and undergo thermal denaturation with a large enthalpy change, mainly contributed by hydration.

Prefoldin is a hexameric molecular chaperone found in the cytosol of archaea and eukaryotes. Its hexameric complex is built from two related classes of subunits, and has the appearance of a jellyfish: Its body consists of a double β-barrel assembly with six long tentacle-like coiled coils protruding from it. Using the tentacles, prefoldin captures an unfolded protein substrate and transfers it to a group II chaperonin. Based on structural information from archaeal prefoldins, mechanisms of substrate recognition and prefoldin-chaperonin cooperation have been investigated. In contrast, the structure and mechanisms of eukaryotic prefoldins remain unknown. In this study, we succeeded in obtaining recombinant prefoldin from a thermophilic fungus, Chaetomium thermophilum (CtPFD). The recombinant CtPFD could not protect citrate synthase from thermal aggregation. However, CtPFD formed a complex with actin from chicken muscle and tubulin from porcine brain, suggesting substrate specificity. We succeeded in observing the complex formation of CtPFD and the group II chaperonin of C. thermophilum (CtCCT) by atomic force microscopy and electron microscopy. These interaction kinetics were analyzed by surface plasmon resonance using Biacore. Finally, we have shown the transfer of actin from CtPFD to CtCCT. The study of the folding pathway formed by CtPFD and CtCCT should provide important information on mechanisms of the eukaryotic prefoldin⁻chaperonin system.

Methylenetetrahydrofolate reductase (MTHFR) is one of the enzymes involved in homocysteine metabolism. Despite considerable genetic and clinical attention, the reaction mechanism and regulation of this enzyme are not fully understood because of difficult production and poor stability. While recombinant enzymes from thermophilic organisms are often stable and easy to prepare, properties of thermostable MTHFRs have not yet been reported.

Hsp104 and its bacterial homolog ClpB form hexameric ring structures and mediate protein disaggregation. The disaggregated polypeptide is thought to thread through the central channel of the ring. However, the dynamic behavior of Hsp104 during disaggregation remains unclear. Here, we reported the stochastic conformational dynamics and a split conformation of Hsp104 disaggregase from Chaetomium thermophilum (CtHsp104) in the presence of ADP by X-ray crystallography, cryo-electron microscopy (EM), and high-speed atomic force microscopy (AFM). ADP-bound CtHsp104 assembles into a 65 left-handed spiral filament in the crystal structure at a resolution of 2.7 Å. The unit of the filament is a hexamer of the split spiral structure. In the cryo-EM images, staggered and split hexameric rings were observed. Further, high-speed AFM observations showed that a substrate addition enhanced the conformational change and increased the split structure's frequency. Our data suggest that split conformation is an off-pathway state of CtHsp104 during disaggregation.

We have developed and characterized a bacterial consortium that reductively dechlorinates trichloroethene to ethene. Quantitative PCR analysis for the 16S rRNA and reductive dehalogenase genes showed that the consortium is highly enriched with Dehalococcoides spp. that have two vinyl chloride reductive dehalogenase genes, bvcA and vcrA, and a trichloroethene reductive dehalogenase gene, tceA. The metagenome analysis of the consortium by the next generation sequencer SOLiD 3 Plus suggests that a Dehalococcoides sp. that is highly homologous to D. mccartyi 195 and equipped with vcrA and tceA exists in the consortium. We isolated this Dehalococcoides sp. and designated it as D. mccartyi UCH-ATV1. As the growth of D. mccartyi UCH-ATV1 is too slow under isolated conditions, we constructed a consortium by mixing D. mccartyi UCH-ATV1 with several other bacteria and performed metagenomic sequencing using the single molecule DNA sequencer PacBio RS II. We successfully determined the complete genome sequence of D. mccartyi UCH-ATV1. The strain is equipped with vcrA and tceA, but lacks bvcA. Comparison with tag sequences of SOLiD 3 Plus from the original consortium shows a few differences between the sequences. This suggests that a genome rearrangement of Dehalococcoides sp. occurred during culture.

Olfactory systems have evolved the extraordinary capability to detect and discriminate volatile odorous molecules (odorants) in the environment. Fundamentally, this process relies on the interaction of odorants and their cognate olfactory receptors (ORs) encoded in the genome. Here, we conducted a cell-based screen using over 800 mouse ORs against seven odorants, resulting in the identification of a set of high-affinity and/or broadly-tuned ORs. We then test whether heterologously expressed ORs respond to odors presented in vapor phase by individually expressing 31 ORs to measure cAMP responses against vapor phase odor stimulation. Comparison of response profiles demonstrates this platform is capable of discriminating between structural analogs. Lastly, co-expression of carboxyl esterase Ces1d expressed in olfactory mucosa resulted in marked changes in activation of specific odorant-OR combinations. Altogether, these results establish a cell-based volatile odor detection and discrimination platform and form the basis for an OR-based volatile odor sensor.

The authentication of honey is important to protect industry and consumers from such adulterated honey. However, until now, there has been no guarantee of honey's authenticity, especially in Indonesia. The classification of honey is based on the bee species (spp.) that produces it. The study used honey from sting bee Apis spp. and stingless bee Tetragonula spp. based on the fact that the content off honey produced between them has differences. Authenticating honey with currently available rapid detection methods, such as 13C nuclear magnetic resonance analysis, is costly. This study aimed to develop an inexpensive, fast, precise, and accurate classification method for authenticating honey.

The kelulut bee (Meliponini) is a subfamily of stingless bees that produce honey. A total of 89 species out of a total of 500 species of kelulut bees are known to originate from the Indo-Australian region. Kelulut bees do not have quality standards so they still refer to the Codex and EU Directive which basically only applied for Apis honey. The Codex and EU Directive are formed by several psychochemical parameters, one of it is diastase activity. Diastase activity in kelulut honey is known not to meet existing standards or even undetectable. Therefore, this study aimed to explore proteins inside kelulut honey and investigate the possibility of using a specific protein as a biomarker to differentiate honey produced by kelulut bee from other honey. This research can also be considered as an initial step to optimize the exploration of protein in kelulut honey. This research is divided into two sections which are the preliminary research and the research expansion. From preliminary section, glucose dehydrogenase enzyme (GDH) was found to be present inside Tetragonula spp honey. A further examination of GDH enzyme was made in four kelulut bee honeys namely Tetragonula leaviceps, T. biroi, Heterotrigona itama, and Geniotrigona thoracica. The preliminary research has five stages that are exactly the as expansion research section except it didn't include GDH activity measurement. The research includes seven main stages. First honeys were dialyzed to remove the sugar content followed by centrifugation. The samples were then purified using liquid chromatography with anion exchanger column. The molecular weight of proteins was analysed by SDS-PAGE method. The GDH activity was measured using spectrophotometer followed by qualitative analysis using LC-MS/MS. The peptide sequences resulted from LC-MS/MS were then matched with Uniprot to identify the unknow protein. The results showed that only T. biroi and T. laeviceps had GDH enzyme activity of 0,1891 U/mL and 0,1652-1,579 U/mL, respectively. Bands from both species were also qualitatively identified as GDH. With these results, it can be concluded that the GDH enzyme cannot be used as a biomarker to distinguish the kelulut honey.

Plasmodium falciparum parasitophorous vacuolar protein 1 (PfPV1), a protein unique to malaria parasites, is localized in the parasitophorous vacuolar (PV) and is essential for parasite growth. Previous studies suggested that PfPV1 cooperates with the Plasmodium translocon of exported proteins (PTEX) complex to export various proteins from the PV. However, the structure and function of PfPV1 have not been determined in detail. In this study, we undertook the expression, purification, and characterization of PfPV1. The tetramer appears to be the structural unit of PfPV1. The activity of PfPV1 appears to be similar to that of molecular chaperones, and it may interact with various proteins. PfPV1 could substitute CtHsp40 in the CtHsp104, CtHsp70, and CtHsp40 protein disaggregation systems. Based on these results, we propose a model in which PfPV1 captures various PV proteins and delivers them to PTEX through a specific interaction with HSP101.

Olfaction is mediated via olfactory receptors (ORs) that are expressed on the cilia membrane of olfactory sensory neurons in the olfactory epithelium. The functional expression of most ORs requires the assistance of receptor-transporting proteins (RTPs). We examined the interactome of RTP1S and OR via proximity biotinylation. Deubiquitinating protein VCIP135, the F-actin-capping protein sub-unit alpha-2, and insulin-like growth factor 2 mRNA-binding protein 2 were biotinylated via AirID fused with OR, RTP1S-AirID biotinylated heat shock protein A6 (HSPA6), and double-stranded RNA-binding protein Staufen homolog 2 (STAU2). Co-expression of HSPA6 partially enhanced the surface expression of Olfr544. The surface expression of Olfr544 increased by 50-80%. This effect was also observed when RTP1S was co-expressed. Almost identical results were obtained from the co-expression of STAU2. The interactions of HSPA6 and STAU2 with RTP1S were examined using a NanoBit assay. The results show that the RTP1S N-terminus interacted with the C-terminal domain of HSP6A and the N-terminal domain of STAU2. In contrast, OR did not significantly interact with STAU2 and HSPA6. Thus, HSP6A and STAU2 appear to be involved in the process of OR traffic through interaction with RTP1S.

Prefoldin is a molecular chaperone that captures an unfolded protein substrate and transfers it to a group II chaperonin. Previous studies have shown that the interaction sites for prefoldin are located in the helical protrusions of group II chaperonins. However, it does not exclude the possibility of the existence of other interaction sites. In this study, we constructed C-terminal truncation mutants of a group II chaperonin and examined the effects of these mutations on the chaperone's function and interaction with prefoldin. Whereas the mutants with up to 6 aa truncation from the C-terminus retained more than 90% chaperone activities for protecting citrate synthase from thermal aggregation and refolding of green fluorescent protein and isopropylmalate dehydrogenase, the truncation mutants showed decreased affinities for prefoldin. Consequently, the truncation mutants showed reduced transfer efficiency of the denatured substrate protein from prefoldin and subsequent chaperonin-dependent refolding. The results clearly show that the C-terminal region of group II chaperonins contributes to their interactions with prefoldin, the transfer of the substrate protein from prefoldin and its refolding.

The NADH oxidase-peroxiredoxin (Prx) system of Amphibacillus xylanus reduces hydroperoxides with the highest turnover rate among the known hydroperoxide-scavenging enzymes. The high electron transfer rate suggests that there exists close interaction between NADH oxidase and Prx. Variant enzyme experiments indicated that the electrons from β-NADH passed through the secondary disulfide, Cys128-Cys131, of NADH oxidase to finally reduce Prx. We previously reported that ionic strength is essential for a system to reduce hydroperoxides. In this study, we analyzed the effects of ammonium sulfate (AS) on the interaction between NADH oxidase and Prx by surface plasmon resonance analysis. The interaction between NADH oxidase and Prx was observed in the presence of AS. Dynamic light scattering assays were conducted while altering the concentration of AS and the ratio of NADH oxidase to Prx in the solutions. The results revealed that the two proteins formed a large oligomeric assembly, the size of which depended on the ionic strength of AS. The molecular mass of the assembly converged at approximately 300 kDa above 240 mM AS. The observed reduction rate of hydrogen peroxide also converged at the same concentration of AS, indicating that a complex formation is required for activation of the enzyme system. That the complex generation is dependent on ionic strength was confirmed by ultracentrifugal analysis, which resulted in a signal peak derived from a complex of NADH oxidase and Prx (300 mM AS, NADH oxidase: Prx = 1:10). The complex formation under this condition was also confirmed structurally by small-angle X-ray scattering.

Olfaction is mediated by the binding of odorant molecules to olfactory receptors (ORs). There are numerous proteins in the nasal mucus, and they contribute to olfaction through various mechanisms. Cytochrome P450 (CYP) family members are known to be present in the olfactory epithelium and are thought to affect olfaction by enzymatic conversion of odorant molecules. In this study, we examined the effects of CYPs on the ligand responses of ORs in heterologous cells. Among the CYPs tested, co-expression of CYP1a2 significantly affected the responses of various ORs, including MOR161-2, to acetophenone. Conversion of acetophenone to methyl salicylate was observed in the medium of CYP1a2-expressing cells. MOR161-2-expressing cells exhibited significantly greater responses to methyl salicylate than to acetophenone. Finally, we analyzed the responses of olfactory neurons expressing MOR161-2 in vivo using the phosphorylated ribosomal protein S6 as a marker. MOR161-2 responded to both acetophenone and methyl salicylate in vivo. When the olfactory mucus was washed out by the injection of PBS to mouse nasal cavity, the response of MOR161-2 to acetophenone was reduced, while that to methyl salicylate did not change. Our data suggest that CYP1a2 affects OR activation by converting acetophenone to methyl salicylate.