Cryo-EM structures of lipidic fibrils of amyloid-ß (1-40).

Alzheimer's disease (AD) is a progressive and incurable neurodegenerative disease characterized by the extracellular deposition of amyloid plaques. Investigation into the composition of these plaques revealed a high amount of amyloid-ß (Aß) fibrils and a high concentration of lipids, suggesting that fibril-lipid interactions may also be relevant for the pathogenesis of AD. Therefore, we grew Aß40 fibrils in the presence of lipid vesicles and determined their structure by cryo-electron microscopy (cryo-EM) to high resolution. The fold of the major polymorph is similar to the structure of brain-seeded fibrils reported previously. The majority of the lipids are bound to the fibrils, as we show by cryo-EM and NMR spectroscopy. This apparent lipid extraction from vesicles observed here in vitro provides structural insights into potentially disease-relevant fibril-lipid interactions.

Determinants of PB1 Domain Interactions in Auxin Response Factor ARF5 and Repressor IAA17.

Auxin is a plant hormone that is central to plant growth and development from embryogenesis to senescence. Auxin signaling is mediated by auxin response transcription factors (ARFs) and Aux/IAA repressors that regulate the expression of a multitude of auxin response genes. ARF and Aux/IAA proteins assemble into homomeric and heteromeric complexes via their conserved PB1 domains. Here we report the first crystal structure of the PB1 complex between ARF5 and IAA17 of Arabidopsis thaliana, which represents the transcriptionally repressed state at low auxin levels. The PB1 domains assemble in a head-to-tail manner with a backbone arrangement similar to that of the ARF5:ARF5 PB1 complex. The ARF5:IAA17 complex, however, reveals distinct points of contact that promote the ARF5:IAA17 interaction over the ARF5:ARF5 interaction. Specifically, surface charges at the interface form salt-bridges that distinguish the homomeric and heteromeric complexes, revealing common and specific interfaces between transcriptionally repressed and derepressed states. Further, the salt-bridges can be reconfigured to switch the affinity between homomeric and heteromeric complexes in an incremental manner. The complex structure combined with quantitative binding analyses would be essential for deciphering the PB1 interaction code underlying the transcriptional regulation of auxin signaling.

Mechanism Underlying Green Discolouration of Myoglobin Induced by Atmospheric Pressure Plasma.

In this study, we elucidated the mechanism underlying atmospheric pressure plasma (APP)-induced green discolouration of myoglobin. Green-coloured pigments are produced upon conversion of myoglobin into sulphmyoglobin, choleglobin, verdoheme, nitrihemin, or nitrimyoglobin. We exposed myoglobin dissolved in phosphate buffer to APP for 20 min and found a decrease in a* value (+redness/-greenness) and increase in b* value (+yellowness/-blueness) (P < 0.05). In the ultraviolet absorption spectrum, myoglobin treated with APP for 20 min showed absorption peaks at 503 and 630 nm, a spectrum different from that of sulphmyoglobin or choleglobin. The secondary structure and molecular weight of myoglobin were unaffected by APP treatment, excluding the possibility of verdoheme or nitrihemin formation. After APP treatment, nitrite was produced in myoglobin solution that provided a positive environment for nitrimyoglobin formation. However, the addition of 0.5% sodium dithionite, a strong reducing agent, to myoglobin solution resulted in the formation of deoxymyoglobin, which was subsequently converted to nitrosomyoglobin upon APP treatment to yield a desirable red colour. Thus, APP-induced green colouration in myoglobin solution is associated with nitrimyoglobin formation. The addition of the antioxidant resulted in the production of red colour in myoglobin solution after APP treatment owing to nitrosomyoglobin formation.

Solution structure and dynamics of anti-CRISPR AcrIIA4, the Cas9 inhibitor.

The bacterial CRISPR-Cas system provides adaptive immunity against invading phages. Cas9, an RNA-guided endonuclease, specifically cleaves target DNA substrates and constitutes a well-established platform for genome editing. Recently, anti-CRISPR (Acr) proteins that inhibit Cas9 have been discovered, promising a useful off-switch for Cas9 to avoid undesirable off-target effects. Here, we report the solution structure and dynamics of Listeria monocytogenes AcrIIA4 that inhibits Streptococcus pyogenes Cas9 (SpyCas9). AcrIIA4 forms a compact monomeric αßßßαα fold comprising three antiparallel ß strands flanked by three α-helices and a short 310-helix. AcrIIA4 exhibits distinct backbone dynamics in fast and slow timescales at loop regions that form interaction surfaces for SpyCas9. In particular, the ß1-ß2 loop that binds to the RuvC domain of SpyCas9 is highly mobile, and the ß1-ß2 and α2-α3 loops that bind to the RuvC and C-terminal domains of SpyCas9, respectively, undergoes conformational exchanges in microsecond-to-millisecond time scales. AcrIIA4 binds to apo-SpyCas9 with KD ~4.8 µM, which compares to KD ~0.6 nM for AcrIIA4 binding to sgRNA-bound SpyCas9. Since the binary complex between AcrIIA4 and SpyCas9 does not compete with the target DNA binding, it can effectively disable the Cas9 nuclease activity by forming a tight ternary complex in the presence of sgRNA.

The transgenic chicken derived anti-CD20 monoclonal antibodies exhibits greater anti-cancer therapeutic potential with enhanced Fc effector functions.

Modern genetic techniques, enable the use of animal bioreactor systems for the production and functional enhancement of anti-cancer antibodies. Chicken is the most efficient animal bioreactor for the production of anti-cancer antibodies because of its relatively short generation time, plentiful reproductive capacity, and daily deposition in the egg white. Although several studies have focused on the production of anti-cancer antibodies in egg white, in-depth studies of the biological activity and physiological characteristics of transgenic chicken-derived anti-cancer antibodies have not been fully carried out. Here, we report the production of an anti-cancer monoclonal antibody against the CD20 protein from egg whites of transgenic hens, and validated the bio-functional activity of the protein in B-lymphoma and B-lymphoblast cells. Quantitative analysis showed that deposition of the chickenised CD20 monoclonal antibody (cCD20 mAb) from transgenic chickens increased in successive generations and with increasing transgene copy number. Ultra-performance liquid chromatography (UPLC) tandem mass spectrometry (LC/MS/MS) analysis showed that the cCD20 mAb exhibited 14 N-glycan patterns with high-mannose, afucosylation and terminal galactosylation. The cCD20 mAb did not exhibit significantly improved Fab-binding affinity, but showed markedly enhanced Fc-related functions, including complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC) compared to commercial rituximab, a chimeric mAb against CD20. Our results suggest that the transgenic chicken bioreactor is an efficient system for producing anti-cancer therapeutic antibodies with enhanced Fc effector functions.

Structural basis for the auxin-induced transcriptional regulation by Aux/IAA17.

Auxin is the central hormone that regulates plant growth and organ development. Transcriptional regulation by auxin is mediated by the auxin response factor (ARF) and the repressor, AUX/IAA. Aux/IAA associates with ARF via domain III-IV for transcriptional repression that is reversed by auxin-induced Aux/IAA degradation. It has been known that Aux/IAA and ARF form homo- and hetero-oligomers for the transcriptional regulation, but what determines their association states is poorly understood. Here we report, to our knowledge, the first solution structure of domain III-IV of Aux/IAA17 (IAA17), and characterize molecular interactions underlying the homotypic and heterotypic oligomerization. The structure exhibits a compact ß-grasp fold with a highly dynamic insert helix that is unique in Aux/IAA family proteins. IAA17 associates to form a heterogeneous ensemble of front-to-back oligomers in a concentration-dependent manner. IAA17 and ARF5 associate to form homo- or hetero-oligomers using a common scaffold and binding interfaces, but their affinities vary significantly. The equilibrium dissociation constants (KD) for homo-oligomerization are 6.6 µM and 0.87 µM for IAA17 and ARF5, respectively, whereas hetero-oligomerization reveals a ∼ 10- to ∼ 100-fold greater affinity (KD = 73 nM). Thus, individual homo-oligomers of IAA17 and ARF5 spontaneously exchange their subunits to form alternating hetero-oligomers for transcriptional repression. Oligomerization is mainly driven by electrostatic interactions, so that charge complementarity at the interface determines the binding affinity. Variable binding affinity by surface charge modulation may effectively regulate the complex interaction network between Aux/IAA and ARF family proteins required for the transcriptional control of auxin-response genes.