Biology subjects

Wu, X. N.

Publications and source records attributed to Wu, X. N..

5 recordsLinked to original sources

bioRxiv2026

An engineered IdeS variant with enhanced activity and performance for IgG degradation

IgG-degrading enzymes have emerged as innovative therapeutic agents for treating conditions driven by pathogenic antibodies. Here, we used structure-guided rational design to engineer IdeSM33, a double mutant (K167R/D226E) of the IgG-specific bacterial protease IdeS from Streptococcus pyogenes, with improved catalytic efficiency. Biolayer interferometry revealed a fourfold increase in binding affinity relative to wild-type IdeS (IdeSWT). This enhancement is likely attributable to mutations that strengthen hydrogen bonding at the enzyme-IgG Fc interface. In vitro, IdeSM33 has higher performance than IdeSWT in cleaving serum IgG. In vivo studies in rabbits demonstrated that IdeSM33 effectively depleted circulating IgG and showed better performance at a dose of 0.005 mg/kg than the IdeSWT. Although doses greater than 0.2 mg/kg demonstrated higher plasma concentrations of IdeS and a larger AUClast, they did not show a significant enhancement in the pharmacodynamics of IgG degradation. Importantly, a single dose of IdeSM33 (0.2 mg/kg) potently degraded binding and neutralizing antibodies against AAV9 within 1-2 days and restored hepatic AAV9 transduction in pre-immunized animals. Together, these findings highlight IdeSM33 as a potent and safe engineered enzyme with therapeutic potential for autoimmune disorders, transplant rejection, and overcoming pre-existing humoral immunity in gene therapy.

bioengineering↗

bioRxiv2022

PEP7 is a ligand for receptor kinase SIRK1 to regulate aquaporins and root growth

Plant receptor kinases constitute a large protein family that regulate various aspects of development and responses to external biotic and abiotic cues. Functional characterization of this protein family and particularly the identification of their ligands remains a major challenge in plant biology. Previously, we identified plasma membrane-intrinsic SUCROSE INDUCED RECEPTOR KINASE 1 (SIRK1) and QIAN SHOU KINASE 1 (QSK1) as a receptor / co-receptor pair involved in regulation of aquaporins in response to osmotic conditions induced by sucrose. Here, we identified a member of the Elicitor Peptide (PEP) family, namely PEP7, as the specific ligand of receptor kinase SIRK1. PEP7 binds to the extracellular domain of SIRK1 with a binding constant of 1.44{+/-}0.79 {micro}M and is secreted to the apoplasm specifically in response to sucrose treatment. Stabilization of a signaling complex involving SIRK1, QSK1 and aquaporins as substrates is mediated by alterations in the external sucrose concentration or by PEP7 application. Moreover, the presence of PEP7 induces the phosphorylation of aquaporins in vivo and enhance water influx into protoplasts. The loss-of-function mutant of SIRK1 is not responsive to external PEP7 treatment regarding kinase activity, aquaporin phosphorylation and water influx activity. Our data indicate that the PEP7/SIRK1/QSK1 complex represents a crucial perception and response module mediating sucrose-controlled water flux in plants.

plant biology↗

bioRxiv2020

A phospho-switch in the N-terminus of NRT2.1 affects nitrate uptake by controlling the interaction of NRT2.1 with NAR2.1

NRT2.1 can be phosphorylated at five different sites within N- and C-terminus. Here, we provide a systematic functional characterization of phosphorylation at S21 and S28 within the N-terminus of NRT2.1. We used existing phosphoproteomic data sets of nitrate starvation and nitrate resupply to construct a site-specific correlation network identifying kinase candidates to phosphorylate NRT2.1. By this approach, we identified NO_SCPCAPITRATEC_SCPCAP UO_SCPCAPPTAKEC_SCPCAP RO_SCPCAPEGULATORYC_SCPCAP KO_SCPCAPINASEC_SCPCAP 1 (AT5G49770) which itself was regulated by phosphorylation at S839 and S870 within its kinase domain. In the active state, when S839 was dephosphorylated and S870 was phosphorylated, NURK1 was found to interact with NRT2.1 at dephosphorylated S28. Upon that interaction, NURK1 can phosphorylate NRT2.1 at S21. Phosphorylation of NRT2.1 at S21 resulted in low interaction of NRT2.1 with its activator protein NAR2.1. By contrast, phosphorylation of NRT2.1 at S28 by a yet unknown kinase enhanced the interaction with NAR2.1, but inhibited the interaction with NURK1. We propose that serines S21 and S28 are involved in a phospho-switch mechanism and by which the interaction of NRT2.1 with its activator NAR2.1, and thus NRT2.1 activity, is modulated. NURK1 here was identified as the kinase affecting this phospho-switch through phosphorylation of NRT2.1 at S21 leading to inactivation of NRT2.1.

plant biology↗

bioRxiv2019

Plasma membrane associated Receptor Like Kinases relocalise to plasmodesmata in response to osmotic stress.

Plasmodesmata act as key elements in intercellular communication, coordinating processes related to plant growth, development and responses to environmental stresses. While many of the developmental, biotic and abiotic signals are primarily perceived at the plasma membrane (PM) by receptor proteins, plasmodesmata also cluster receptor-like activities and whether or not these two pathways interact is currently unknown.\n\nHere we show that specific PM-located Leucine-Rich-Repeat Receptor-Like-Kinases (LRR-RLKs), KIN7 and IMK2, which under optimal growth conditions are absented from plasmodesmata, rapidly relocate and cluster to the pores in response to osmotic stress. This process is remarkably fast, it is not a general feature of PM-associated proteins and is independent of sterol- and sphingolipid-membrane composition. Focusing on KIN7, previously reported to be involved in stress responses, we show that relocalisation upon mannitol depends on KIN7 phosphorylation. Loss-of-function mutation in KIN7 induces delay in lateral root (LR) development and the mutant is affected in the root response to mannitol stress. Callose-mediated plasmodesmata regulation is known to regulate LR development. We found that callose levels are reduced in kin7 mutant background with a root phenotype resembling ectopic expression of PdBG1, an enzyme that degrades callose at the pores. Both the LR and callose phenotypes can be complemented by expression of KIN7 -wild-type and -phosphomimic variants but not by KIN7 phosphodead mutant which fails to relocalise at plasmodesmata. Together the data indicate that re-organisation of RLKs to plasmodesmata is important for the regulation of callose and LR development as part of the plant response to osmotic stress.

plant biology↗

bioRxiv2019

The systemin signaling cascade as derived from phosphorylation time courses under stimulation by systemin and its inactive Thr17Ala (A17) analog

Systemin is a small peptide with important functions in plant wound response signaling. Although transcriptional responses of systemin action are well described, the precise signaling cascades involved in its perception and signal transduction are poorly understood at the protein level. Here we use a phosphoproteomic profiling study involving stimulation time courses with systemin and its inactive analogon A17 to reconstruct a systemin-specific kinase/phosphatase signaling network. The time course analysis of systemin-induced phosphorylation patterns revealed early events at the plasma membrane, such dephosphorylation of H+-ATPase, rapid phosphorylation of NADPH-oxidase and Ca2+-ATPase. Later responses involved transient phosphorylation of small GTPases and vesicle trafficking proteins, as well as transcription factors. Based on a correlation analysis of systemin-specific phosphorylation profiles, we predict substrate candidates for 56 systemin specific kinases and 18 phosphatases. Among the kinases are several systemin-specific receptor kinases as well as kinases with downstream signaling functions, such as MAP-kinases. A regulatory circuit for plasma membrane H+-ATPase was predicted and confirmed by in-vitro activity assays. In this regulatory model we propose that upon systemin treatment, H+-ATPase LHA1 is rapidly de-phosphorylated at its C-terminal regulatory residue T955 by phosphatase PLL5, resulting in the alkalization of the growth medium within 2 minutes of systemin treatment. We further propose that the H+-ATPase LHA1 is re-activated by MAP-Kinase MPK2 later in the systemin response. MPK2 was identified with increased phosphorylation at its activating TEY-motif at 15 minutes of treatment and the predicted interaction with LHA1 was confirmed by in-vitro kinase assays. Our data set provides a valuable resource of proteomic events involved in the systemin signaling cascade with a focus on predictions of substrates to systemin-specific kinases and phosphatases.

plant biology↗