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Rice straighthead disease substantially reduces crop yield, posing a significant threat to global food security. Dimethylarsinic acid (DMA) is the causal agent of straighthead disease and is highly toxic to the reproductive tissue of rice. However, the precise physiological mechanism underlying DMA toxicity remains unknown. In this study, six rice varieties with varying susceptibility to straighthead were utilized to investigate the growth performance and element distribution in rice panicles under DMA stress through pot experiments, as well as to explore the physiological response to DMA using transcriptomic methods. The findings demonstrate significant variations in both DMA accumulation and straighthead sensitivity among cultivars. The susceptible varieties exhibited higher DMA accumulation indices and displayed typical symptoms of straighthead disease, including erect panicles, deformed rachides and husks, and reduced seed setting rate and grain yield when compared to the resistant varieties. Moreover, DMA addition promoted mineral nutrients to accumulate in rachides and husks but less in grains. DMA showed preferential accumulation in rice grains with a distribution pattern similar to that of Copper (Cu) and zinc (Zn) within the panicle. Transcriptome analyses underscored the substantial impact of DMA on gene expression related to mineral metabolism. Notably, DMA addition significantly up‐regulated the expression of pectin methylesterase, pectin lyase, polygalacturonase, and exogalacturonase genes in Nanjingxiangzhan, while these genes were down‐regulated or weakly expressed in Ruanhuayou 1179. The alteration of pectin metabolic pathways induced by DMA may lead to abnormality of cell wall assembly and modification, thereby resulting in deformed rice panicles. [ABSTRACT FROM AUTHOR]

Death receptor 3 (DR3) (a.k.a. tumor necrosis factor receptor superfamily 25) plays a key role in the immune system by activating nuclear factor kappa‐light‐chain‐enhancer of activated B cells signaling pathway. Here we present the crystal structures of human and mouse DR3 intracellular death domain (DD) at 2.7 and 2.5 Å resolutions, respectively. The mouse DR3 DD adopts a classical six‐helix bundle structure while human DR3 DD displays an extended fold. Though there is one‐amino‐acid difference in the linker between maltose‐binding protein (MBP) tag and DR3 DD, according to our self‐interaction analysis, the hydrophobic interface discovered in MBP–hDR3 DD crystal structure is responsible for both hDR3 DD and mDR3 DD homotypic interaction. Furthermore, our biochemical analysis indicates that the sequence variation between human and mouse DR3 DD does not affect its structure and function. Small‐angle X‐ray scattering analysis shows the averaged solution structures of both human and mouse MBP‐DR3 DD are the combination of different conformations with different proportion. Through switching to the open conformation, DR3 DD could improve the interaction with downstream element TNFR‐associated death domain (TRADD). Here we propose an activation‐dependent structural rearrangement model: the DD region is folded as the six‐helix bundles in the resting state, while upon extracellular ligand engagement, it switches to the open conformation, which facilitates its self‐association and the recruitment of TRADD. Our results provide detailed insights into the architecture of DR3 DD and the molecular mechanism of activation. Databases: All refined structure coordinates as well as the corresponding structure factors have been deposited in the PDB under the accession codes 5YGS, 5YEV, 5YGP, 5ZNY, 5ZNZ. [ABSTRACT FROM AUTHOR]

The zebrafish genome encodes homologs for most of the proteins involved in inflammatory pathways; however, the molecular components and activation mechanisms of fish inflammasomes are largely unknown. ASC [apoptosis‐associated speck‐like protein containing a caspase‐recruitment domain (CARD)] is the only adaptor involved in the formation of multiple types of inflammasomes. Here, we demonstrate that zASC is also involved in inflammasome activation in zebrafish. When overexpressed in vitro and in vivo in zebrafish, both the zASC and zASC pyrin domain (PYD) proteins form speck and filament structures. Importantly, the crystal structures of the N‐terminal PYD and C‐terminal CARD of zebrafish ASC were determined independently as two separate entities fused to maltose‐binding protein. Structure‐guided mutagenesis revealed the functional relevance of the PYD hydrophilic surface found in the crystal lattice. Finally, the fish caspase‐1 homolog Caspy, but not the caspase‐4/11 homolog Caspy2, interacts with zASC through homotypic PYD–PYD interactions, which differ from those in mammals. These observations establish the conserved and unique structural/functional features of the zASC‐dependent inflammasome pathway. Database: Structural data are available in the PDB under accession numbers 5GPP and 5GPQ. [ABSTRACT FROM AUTHOR]

Recent outbreak of flavivirus Zika virus (ZIKV) in America has urged the basic as well as translational studies of this important human pathogen. The nonstructural protein 5 (NS5) of the flavivirus has an N-terminal methyltransferase (MTase) domain that plays critical roles in viral RNA genome capping. The null mutant of NS5 MTase is lethal for virus. Therefore, NS5 is a potential drug target for the treatment of Zika virus infection. In this study, we determined crystal structures of the ZIKV MTase in complex with GTP and RNA cap analogue 7me GpppA. Structural analyses revealed highly conserved GTP/cap-binding pocket and S-adenosylmethionine (SAM)-binding pocket. Two conformations of the second base of the cap were identified, which suggests the flexibility of RNA conformation. In addition, the ligand-binding pockets identified a continuous region of hotspots suitable for drug design. Docking calculation shows that the Dengue virus inhibitor compound 10 may bind to the ZIKV MTase. [ABSTRACT FROM AUTHOR]

Crystallins are structural proteins in the lens that last a lifetime with little turnover. Deviant in crystallins can cause rare but severe visual impairment, namely, congenital cataracts. It is reported that several mutations in the acidic β-crystallin 4 (CRYBA4) are related to congenital cataracts. However, the pathogenesis of these mutants is not well understood at molecular level. Here we evaluate the biochemical properties of wild type CRYBA4 (CRYBA4WT) and a pathogenic G64W mutant (CRYBA4G64W) including protein folding, polymerization state and protein stability. Furthermore, we explore the differences in their interactions with α-crystallin A (CRYAA) and basic β-crystallin 1 (CRYBB1) via yeast two-hybrid and pull-down assay in vitro , through which we find that G64W mutation leads to protein misfolding, decreases protein stability, blocks its interaction with CRYBB1 but maintains its interaction with CRYAA. Our results deepen our understanding of the pathogenesis of congenital cataracts. • The congenital cataract causing mutation (c.225G > T, p.G64W) leads to misfolding of acidic β-crystallin 4 protein. • The stability of acidic β-crystallin 4 is affected by Gly64 to Trp mutation. • Acidic β-crystallin 4 loses its interaction with basic β-crystallin 1 due to the mutation. • α-Crystallin A fails in helping the mutated protein folding. [ABSTRACT FROM AUTHOR]

The NLRP1 inflammasome functions as canonical cytosolic sensor in response to intracellular infections and is implicated in auto-inflammatory diseases. But the regulation and signal transduction mechanisms of NLRP1 are incompletely understood. Here, we show that the T60 variant of CARD8, but not the canonical T48 isoform, negatively regulates the NLRP1 inflammasome activation by directly interacting with the receptor molecule NLRP1 and inhibiting inflammasome assembly. Furthermore, our results suggest that different ASC preference in three types of inflammasomes, namely the ASC-indispensable NLRP1 inflammasome, ASC-dispensable mNLRP1b inflammasome and ASC-independent CARD8 inflammasome, is mainly caused by the CARD domain, not the UPA subdomain. Based on the systematic site-directed mutagenesis and structural analysis, we find that signal transduction of the NLRP1 inflammasome relies on multiple interaction surfaces at its CARD domain. Finally, our results partly explain how mutations in NLRP1 lead to its constitutive activation in auto-inflammatory diseases. In conclusion, our study not only reveals how CARD8 downregulates the NLRP1 inflammasome activation, but also provides insights into the assembly mechanisms of CARD-containing inflammasomes., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Xu, Deng, Huang, Yang, Sun, Liu, Zhao, Zeng, Yin, Zheng, Wang, Liu, Zhao, Xiao, Zhou and Jin.)

Background: Death domain superfamily are compact structural motifs that play critical roles in many biological processes including inflammation and apoptosis. Due to mediating protein-protein interactions, these death domains have a high tendency to form oligomers and are notoriously difficult to deal with when overexpressed in vitro. In this study, we found that maltose binding protein (MBP) is very effective in improving the behavior of many death domain superfamily members., Methods: In order to achieve high-level expression of death domain superfamily in E. coli, we designed two MBPtagged expression vectors based on a pET30a backbone: one with a short flexible noncleavable linker, the other with a TEV cleavage site. Soluble protein was purified from cell lysate by HisTrapTM IMAC column and Superdex-200 gel filtration column., Results: We tested seven targets of death domain superfamily, including two PYDs, three CARDs, and two DDs; for all of these, it is challenging to obtain recombinant protein without a tag. We expressed and purified these death domain proteins successfully as soluble and highly purified protein with both vectors. Among them, two proteins were crystallized successfully., Conclusions: Our study demonstrated that both recombinant MBP expression vectors significantly enhanced production. In addition, MBP tagged recombinant proteins can sometimes produce crystals. This strategy may be applied to other challenging proteins.