The LRR receptor-like kinase ALR1 is a plant aluminum ion sensor.

Plant survival requires an ability to adapt to differing concentrations of nutrient and toxic soil ions, yet ion sensors and associated signaling pathways are mostly unknown. Aluminum (Al) ions are highly phytotoxic, and cause severe crop yield loss and forest decline on acidic soils which represent ∼30% of land areas worldwide. Here we found an Arabidopsis mutant hypersensitive to Al. The gene encoding a leucine-rich-repeat receptor-like kinase, was named Al Resistance1 (ALR1). Al ions binding to ALR1 cytoplasmic domain recruits BAK1 co-receptor kinase and promotes ALR1-dependent phosphorylation of the NADPH oxidase RbohD, thereby enhancing reactive oxygen species (ROS) generation. ROS in turn oxidatively modify the RAE1 F-box protein to inhibit RAE1-dependent proteolysis of the central regulator STOP1, thus activating organic acid anion secretion to detoxify Al. These findings establish ALR1 as an Al ion receptor that confers resistance through an integrated Al-triggered signaling pathway, providing novel insights into ion-sensing mechanisms in living organisms, and enabling future molecular breeding of acid-soil-tolerant crops and trees, with huge potential for enhancing both global food security and forest restoration.

Optimization strategies for expression of a novel bifunctional anti-PD-L1/TGFBR2-ECD fusion protein.

The novel anti-PD-L1/TGFBR2-ECD fusion protein (BR102) comprises an anti-PD-L1 antibody (HS636) which is fused at the C terminus of the heavy chain to a TGF-ß1 receptor Ⅱ ectodomain (TGFBR2-ECD), and which can sequester the PD-1/PD-L1 pathway and TGF-ß bioactivity in the immunosuppressive tumor microenvironment. In the expression of TGFBR2-ECD wild-type fused protein (BR102-WT), a 50 kDa clipped species was confirmed to be induced by proteolytic cleavage at a "QKS" site located in the N-terminus of the ectodomain, which resulted in the formation of IgG-like clipping. The matrix metalloproteinase-9 was determined to be associated with BR102-WT digestion. In addition, it was observed that the N-glycosylation modifications of the fusion protein were tightly involved in regulating proteolytic activity and the levels of cleavage could be significantly suppressed by MMP-inhibitors. To avoid proteolytic degradation, eliminating protease-sensitive amino acid motifs and introducing potential glycosylation were performed. Three sensitive motifs were mutated, and the levels of clipping were strongly restrained. The mutant candidates exhibited similar binding affinities to hPD-L1 and hTGF-ß1 as well as highly purified BR102-WT2. Furthermore, the mutants displayed more significant proteolytic resistance than that of BR102-WT2 in the lysate incubation reaction and the plasma stability test. Moreover, the bifunctional candidate Mu3 showed an additive antitumor effect in MC38/hPD-L1 bearing models as compared to that of with anti-PD-L1 antibody alone. In conclusion, in this study, the protease-sensitive features of BR102-WT were well characterized and efficient optimization was performed. The candidate BR102-Mutants exhibited advanced druggability in drug stability and displayed desirable antitumor activity.

Isolation, physicochemical, and structure-function relationship of the hydrophobic variant of Fc-fusion proteins that bind to TNF-α receptor, HS002 and HS002A.

HS002 is the recombinant human tumor necrosis factor-α receptor Ⅱ: IgG Fc fusion protein licensed in China to treat rheumatism and psoriasis. The aim of this study was to isolate and characterize the hydrophobic freeze-dried powder injection (HS002) and ampoule injection (HS002A) variants derived from proteins of the same sequence and then to explore the structure-function relationship. Extensive physicochemical and structural testing was performed during a side-by-side comparison of the monomer peak and variant. Then the TNF-α-related binding activity, cell biological activity and affinity with FcRn were analyzed. Finally, a transformation study of the hydrophobic variant was performed under serum-like redox conditions. This research revealed that HS002A has similar physicochemical and structure-function relationship profiles to those of HS002. The hydrophobic variant exhibited the presence of new incorrect disulfide bridging. At the same time, this novel disulfide scrambled species structure-function relationship was found to be the molecular basis for reduced TNF-α binding and cell biological activities. In addition, incorrect disulfide bridging was found to be reversible under serum-like redox conditions, restoring TNF-α binding and cell biological activities to almost normal levels, all of which indicate that the variant is probably irrelevant to clinical efficacy once the drug enters the bloodstream.

A novel twin-column continuous chromatography approach for separation and enrichment of monoclonal antibody charge variants.

Downstream processing of mAb charge variants is difficult owing to their similar molecular structures and surface charge properties. This study aimed to apply a novel twin-column continuous chromatography (called N-rich mode) to separate and enrich acidic variants of an IgG1 mAb. Besides, a comparison study with traditional scaled-up batch-mode cation exchange (CEX) chromatography was conducted. For the N-rich process, two 3.93 mL columns were used, and the buffer system, flow rate and elution gradient slope were optimized. The results showed that 1.33 mg acidic variants with nearly 100% purity could be attained after a 22-cycle accumulation. The yield was 86.21% with the productivity of 7.82 mg/L/h. On the other hand, for the batch CEX process, 4.15 mL column was first used to optimize the separation conditions, and then a scaled-up column of 88.20 mL was used to separate 1.19 mg acidic variants with the purity of nearly 100%. The yield was 59.18% with the productivity of 7.78 mg/L/h. By comparing between the N-rich and scaled-up CEX processes, the results indicated that the N-rich method displays a remarkable advantage on the product yield, i.e. 1.46-fold increment without the loss of productivity and purity. Generally, twin-column N-rich continuous chromatography displays a high potential to enrich minor compounds with a higher yield, more flexibility and lower resin cost.

Ethylene promotes seed iron storage during Arabidopsis seed maturation via ERF95 transcription factor.

Because Iron (Fe) is an essential element, Fe storage in plant seeds is necessary for seedling establishment following germination. However, the mechanisms controlling seed Fe storage during seed development remain largely unknown. Here we reveal that an ERF95 transcription factor regulates Arabidopsis seed Fe accumulation. We show that expression of ERF95 increases during seed maturation, and that lack of ERF95 reduces seed Fe accumulation, consequently increasing sensitivity to Fe deficiency during seedling establishment. Conversely, overexpression of ERF95 has the opposite effects. We show that lack of ERF95 decreases abundance of FER1 messenger RNA in developing seed, which encodes Fe-sequestering ferritin. Accordingly, a fer1-1 loss-of-function mutation confers reduced seed Fe accumulation, and suppresses ERF95-promoted seed Fe accumulation. In addition, ERF95 binds to specific FER1 promoter GCC-boxes and transactivates FER1 expression. We show that ERF95 expression in maturing seed is dependent on EIN3, the master transcriptional regulator of ethylene signaling. While lack of EIN3 reduces seed Fe content, overexpression of ERF95 rescues Fe accumulation in the seed of ein3 loss-of-function mutant. Finally, we show that ethylene production increases during seed maturation. We conclude that ethylene promotes seed Fe accumulation during seed maturation via an EIN3-ERF95-FER1-dependent signaling pathway.

Prdx1 Reduces Intracerebral Hemorrhage-Induced Brain Injury via Targeting Inflammation- and Apoptosis-Related mRNA Stability.

RNA-binding proteins (RBPs) have been shown to be involved in posttranscriptional regulation, which plays an important role in the pathophysiology of intracerebral hemorrhage (ICH). Peroxiredoxin 1 (Prdx1), an RBP, plays an important role in regulating inflammation and apoptosis. On the basis that inflammation and apoptosis may contribute to ICH-induced brain injury, in this study, we used ICH models coupled with in vitro experiments, to investigate the role and mechanism of Prdx1 in regulating inflammation and apoptosis by acting as an RBP after ICH. We first found that Prdx1 was significantly up-regulated in response to ICH-induced brain injury and was mainly expressed in astrocytes and microglia in ICH rat brains. After overexpressing Prdx1 by injecting adeno-associated virus (AAV) into the striatum of rats at 3 weeks, we constructed ICH models and found that Prdx1 overexpression markedly reduced inflammation and apoptosis after ICH. Furthermore, RNA immunoprecipitation combined with high-throughput sequencing (RIP-seq) in vitro revealed that Prdx1 affects the stability of inflammation- and apoptosis-related mRNA, resulting in the inhibition of inflammation and apoptosis. Finally, overexpression of Prdx1 significantly alleviated the symptoms and mortality of rats subjected to ICH. Our results show that Prdx1 reduces ICH-induced brain injury by targeting inflammation- and apoptosis-related mRNA stability. Prdx1 may be an improved therapeutic target for alleviating the brain injury caused by ICH.