Functionalized polysaccharides with aminoguaiacol: a competition between associative behavior and antibacterial and antioxidant activities

In a previous study, we presented the development of a series of functionalized carboxymethylpullulan (CMP) grafted with aminoguaiacol (derivative of guaiacol with known antibacterial and antioxidant activities) leading to CMP-G derivatives with various degrees of substitution [DS(Ga)] from 0.16 to 0.58. Our results have shown the efficiency of the grafting both with the evidence of antioxidant and antibacterial activities (Staphylococcus aureus) of the CMP-G derivatives. Nevertheless, an important result has shown surprisingly that such biological activity was not clearly improved with the DS(Ga) unlike the antioxidant activity. These results were probably correlated with a peculiar associative behavior of the derivative (i.e. amphiphilic character) due to the grafted hydrophobic guaiacol groups leading to preferential intramolecular association which was particularly important in the more concentrated regime (polysoap behavior). To complete this study, we propose here two strategies in order to diminish the associative character and notably the polysoap behavior: (i) decrease the DS(Ga) of CMP derivative with a CMP-G0.05 [grafted with a DS(Ga) = 0.05], (ii) conduct the functionalization onto a more rigid polysaccharide backbone as alginate. Our results show a good correlation of the associative physicochemical behaviors with both antioxidant and antibacterial activities. They also confirm the availability of these strategies mainly for the first one (i.e. CMP-G0.05). The main result indicates that the lower is the DS(Ga), the better is the antibacterial activity thanks to a lower associative character. Finally, this study also shows that the grafting of aminoguaiacol is possible onto another anionic polysaccharide (i.e. alginate).

Oligomerization of NLR immune receptor RPP7 triggered by atypical resistance protein RPW8/HR as ligand

In certain plant hybrids, autoimmunity is triggered by immune components that interact in the absence of a pathogen trigger. Often, NLR immune receptors are involved, with a particularly interesting case in Arabidopsis thaliana involving variants of the NLR RPP7 as well as variants of RPW8/HR proteins, which are homologs of animal MLKL and fungal HELL domain proteins. We demonstrate that HR4Fei-0 but not the closely related HR4Col-0 protein directly disrupts intramolecular association of RPP7bLerik1-3, which in turn initiates P-loop dependent NLR signaling. In agreement, RPP7bLerik1-3 forms a higher-order complex only in the presence of HR4Fei-0 but not HR4Col-0. In addition, we find that HR4Fei-0 on its own can form detergent-resistant oligomers suggestive of amyloid-like aggregates, which in turn can directly kill cells in an RPP7bLerik1-3-independent manner. Our work provides in vivo biochemical evidence for a plant resistosome complex and the mechanisms by which RPW8/HR proteins trigger cell death.

Structural insights into the functional versatility of an FHA domain protein in mycobacterial signaling

Forkhead-associated (FHA) domains are modules that bind to phosphothreonine (pThr) residues in signaling cascades. The FHA-containing mycobacterial protein GarA is a central element of a phosphorylation-dependent signaling pathway that redirects metabolic flux in response to amino acid starvation or cell growth requirements. GarA acts as a phosphorylation-dependent ON/OFF molecular switch. In its nonphosphorylated ON state, the GarA FHA domain engages in phosphorylation-independent interactions with various metabolic enzymes that orchestrate nitrogen flow, such as 2-oxoglutarate decarboxylase (KGD). However, phosphorylation at the GarA N-terminal region by the protein kinase PknB or PknG triggers autoinhibition through the intramolecular association of the N-terminal domain with the FHA domain, thus blocking all downstream interactions. To investigate these different FHA binding modes, we solved the crystal structures of the mycobacterial upstream (phosphorylation-dependent) complex PknB-GarA and the downstream (phosphorylation-independent) complex GarA-KGD. Our results show that the phosphorylated activation loop of PknB serves as a docking site to recruit GarA through canonical FHA-pThr interactions. However, the same GarA FHA–binding pocket targets an allosteric site on nonphosphorylated KGD, where a key element of recognition is a phosphomimetic aspartate. Further enzymatic and mutagenesis studies revealed that GarA acted as a dynamic allosteric inhibitor of KGD by preventing crucial motions in KGD that are necessary for catalysis. Our results provide evidence for physiological phosphomimetics, supporting numerous mutagenesis studies using such approaches, and illustrate how evolution can shape a single FHA-binding pocket to specifically interact with multiple phosphorylated and nonphosphorylated protein partners.

Structure of phosphorylated UBL domain and insights into PINK1-orchestrated parkin activation

Mutations in PARK2 and PARK6 genes are responsible for the majority of hereditary Parkinson’s disease cases. These genes encode the E3 ubiquitin ligase parkin and the protein kinase PTEN-induced kinase 1 (PINK1), respectively. Together, parkin and PINK1 regulate the mitophagy pathway, which recycles damaged mitochondria following oxidative stress. Native parkin is inactive and exists in an autoinhibited state mediated by its ubiquitin-like (UBL) domain. PINK1 phosphorylation of serine 65 in parkin’s UBL and serine 65 of ubiquitin fully activate ubiquitin ligase activity; however, a structural rationale for these observations is not clear. Here, we report the structure of the phosphorylated UBL domain from parkin. We find that destabilization of the UBL results from rearrangements to hydrophobic core packing that modify its structure. Altered surface electrostatics from the phosphoserine group disrupt its intramolecular association, resulting in poorer autoinhibition in phosphorylated parkin. Further, we show that phosphorylation of both the UBL domain and ubiquitin are required to activate parkin by releasing the UBL domain, forming an extended structure needed to facilitate E2–ubiquitin binding. Together, the results underscore the importance of parkin activation by the PINK1 phosphorylation signal and provide a structural picture of the unraveling of parkin’s ubiquitin ligase potential.

Unimolecular micelles from graft copolymer with binary side chains

A novel amphiphilic binary graft copolymer was synthesized and used to prepare unimolecular micelles by intramolecular association.