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Enzymes with a protein kinase fold transfer phosphate from adenosine 5′-triphosphate (ATP) to substrates in a process known as phosphorylation. Here, we show that the Legionella meta-effector SidJ adopts a protein kinase fold, yet unexpectedly catalyzes protein polyglutamylation. SidJ is activated by host-cell calmodulin to polyglutamylate the SidE family of ubiquitin (Ub) ligases. Crystal structures of the SidJ-calmodulin complex reveal a protein kinase fold that catalyzes ATP-dependent isopeptide bond formation between the amino group of free glutamate and the γ-carboxyl group of an active-site glutamate in SidE. We show that SidJ polyglutamylation of SidE, and the consequent inactivation of Ub ligase activity, is required for successful Legionella replication in a viable eukaryotic host cell.

Synthetic polypeptides, also known as poly(α-amino acids), have the same polyamide backbone structures as natural proteins and peptides. As an important class of biomaterials, polypeptides have been widely used because of their biocompatibility, bioactivity and biodegradability. Ring-opening polymerization of N -carboxyanhydride (NCA) is a classical and widely used method for the synthesis of polypeptides. The dominantly used primary amine–initiated NCA polymerization can yield well-defined polymers and complex macromolecular architectures, but the reaction is slow and sensitive to moisture, making it necessary to use anhydrous solvents and a glovebox. One solution is to use lithium hexamethyldisilazide (LiHMDS) as the initiator, as described in this protocol. LiHMDS-initiated NCA polymerization is less sensitive to moisture and can be carried out in an open vessel outside the glovebox. It is also very fast; the reaction can be complete within 5 min to produce 30-mer polypeptides. In this protocol, poly(γ-benzyl- l -glutamate) is prepared as an example, but the protocol can easily be adapted to the synthesis of other polypeptides by generating NCAs from different amino acids, making it particularly suitable for the efficient parallel synthesis of polypeptide libraries. We provide detailed procedures for NCA synthesis and purification, the method of polymer end-group modification and measurement of polymerization kinetics and reactivity ratio. The procedure for synthesis of monomers and polymerization to form polypeptides requires <1 d. The superfast and open-vessel NCA polymerization method described here will probably enable a wide range of applications in the synthesis and functional study of polypeptide biomaterials. Key points Most methods for making synthetic polypeptides require strict anhydrous conditions and long reaction times. The ring-opening polymerization reaction described in this protocol uses N -carboxyanhydride versions of the amino acid monomers and is initiated by using lithium hexamethyldisilazide. This reaction has a mechanism different from that in similar reactions initiated by primary amines and is both faster and less sensitive to moisture. These features make it attractive for the preparation of polypeptide libraries. Synthetic polypeptides are used in many applications in which biocompatibility is important. This protocol describes a fast lithium hexamethyldisilazide-initiated N -carboxyanhydride ring-opening polymerization method for their synthesis.

In response to the limitations of hyaluronic acid (HA)—such as its high cost, short durability, and instability—in anti-aging aesthetic applications, this study developed a novel injectable micelle system, with a triple network structure. It is the particle size of approximately 400 nm and the elevated potential that enhance the crosslinking density and mechanical strength of the hydrogel. Importantly, following ultrafiltration and purification processes, the material’s hemolysis rate measured by spectrophotometry was only 3.25%, and endotoxin levels measured by the LAL assay were less than 0.5 EU/mL (test conditions: 37 °C, pH = 7, detection limit: 0.125 EU/mL). Building on this safe and stable material platform, we further designed an antibacterial wound dressing by functionalizing γ-PGA with penicillin or benzalkonium chloride. It reduced the cellular activity of Staphylococcus aureus by 78.9% and 84.2%, respectively. The outstanding safety profile, combined with customizable functionality, positions this γ-PGA-based platform as a promising multifunctional biomaterial meeting practical standards for both aesthetic medicine and wound care applications.

The impact of chirality on immune response has attracted great interest in cancer vaccine research recently. However, the study of chiral synthetic polypeptide hydrogels as cancer vaccines as well as of the impact of biomaterials themselves for antitumor immunotherapy has rarely been reported. Here, we show the key role of residue chirality of polypeptide hydrogels in antitumor immunity and local immune microenvironment regulation. Compared to poly(γ-ethyl-L-glutamate)-based hydrogels (L-Gel), poly(γ-ethyl-D-glutamate)-based hydrogels (D-Gel) induces enhanced level of immune cell infiltration. However, D-Gel causes higher levels of suppressive markers on antigen-presenting cells and even induces stronger T cell exhaustion than L-Gel. Finally, D-Gel establishes a local chronic inflammatory and immunosuppressive microenvironment and shows insufficient anti-tumor effects. Conversely, the milder host immune responses induced by L-Gel leads to more effective tumor inhibition. This study provides insights on the role of residue chirality in the regulation of local immune microenvironment and affecting antitumor immune response. The impact of chemical chirality on immune response attracts attention in cancer vaccine design recently. Here this group reports the chirality of poly(γ-ethyl-D-glutamate)-based hydrogel exhibiting higher levels of suppression on antigen-presenting cells and inducing stronger T cell exhaustion than L-Gel eventually leading to insufficient anti-tumor efficacy.

Poly-γ-glutamic acid (γ-PGA) is a natural, biodegradable and water-soluble biopolymer of glutamic acid. This review is focused on nonrecombinant microbial production of γ-PGA via fermentation processes. In view of its commercial importance, the emphasis is on l -glutamic acid independent producers (i.e. microorganisms that do not require feeding with the relatively expensive amino acid l -glutamic acid to produce γ-PGA), but glutamic acid dependent production is discussed for comparison. Strategies for improving production, reducing costs and using renewable feedstocks are discussed.

Poly-γ-glutamic acid (γ-PGA) is widely used in the field of biomedicine, food, agriculture, and ecological remediation. For the biosynthesis of γ-PGA, the pigments and remaining glutamate are two big problems that impede γ-PGA production by fermentation, and a trade-off between the decolorization rate and γ-PGA recovery rate during the purification process was found. The optimized static activated carbon adsorption conditions for treating the 2-times diluted cell-free supernatant (i.e., feed solution) was as follows: 0.51% 200-mesh, 1000 iodine value, coal-based activated carbon, pH 6.0, 140 min, and 40 °C. Under the optimized conditions, the decolorization rate reached 94.42%, and the recovery rate of γ-PGA was 94.22%. During the adsorption process, the pigments were adsorbed on the activated carbon surface in a monolayer, and the process was a spontaneous, heat-absorbing, and entropy-increasing process. Then, the decolorization flow rate optimized for the dynamic decolorization experiment was 1 BV/h. However, the remaining glutamate was still a problem after the activated carbon adsorption. After isoelectric point (IEP) precipitation of glutamic acid, the glutamic acid can be recovered, and the residual pigment can be further removed. Finally, an integrated decolorization process of activated carbon adsorption and IEP precipitation of glutamic acid was developed. After the integrated process, the decolorization and glutamic acid precipitation rates were 95.80% and 49.02%, respectively. The recovered glutamic acid can be reused in the next fermentation process.

A novel Bacillus subtilis HB-31 strain was isolated from Gotjawal Wetland in Jeju Island, Republic of Korea. A mucus substance produced by this strain was identified as high-molecular-weight poly-γ-glutamic acid (γ-PGA) using NMR, Fourier transform infrared spectroscopy, and size-exclusion chromatography/multi-angle light scattering analyses. We evaluated whether γ-PGA strengthened the skin barrier using keratinocytes and a reconstructed skin model. In keratinocytes, γ-PGA treatment dose-dependently increased the mRNA expression of skin barrier markers, including filaggrin, involucrin, loricrin, serine palmitoyl transferase, fatty acid synthase, and 3-hydroxy-3-methylglutaryl coenzyme A reductase. γ-PGA also enhanced hyaluronic acid synthesis by upregulating hyaluronic acid synthase-1, -2, and -3 mRNA levels and promoted aquaporin 3 expression, which is involved in skin hydration. In the reconstructed skin model, topical application of 1% γ-PGA elevated filaggrin, involucrin, CD44, and aquaporin 3 expression, compared to the control. These results suggest that the newly isolated HB-31 can be used as a commercial production system of high-molecular-weight γ-PGA, which can serve as an effective ingredient for strengthening the skin barrier and improving moisture retention. Further research is needed to explore the long-term effects of γ-PGA on skin health and its application in treating skin conditions.

In recent years, there has been an increasing interest in oncolytic adenoviral vectors as an alternative anticancer therapy. The induction of an immune response can be considered as a major limitation of this kind of application. Significant research efforts have been focused on the development of biodegradable polymer poly-gamma-glutamic acid (γ-PGA)-based nanoparticles used as a vector for effective and safe anticancer therapy, owing to their controlled and sustained-release properties, low toxicity, as well as biocompatibility with tissue and cells. This study aimed to introduce a specific destructive and antibody blind polymer-coated viral vector into cancer cells using γ-PGA and chitosan (CH). Adenovirus was successfully encapsulated into the biopolymer particles with an encapsulation efficiency of 92% and particle size of 485 nm using the ionic gelation method. Therapeutic agents or nanoparticles (NPs) that carry therapeutics can be directed specifically to cancerous cells by decorating their surfaces using targeting ligands. Moreover, in vitro neutralizing antibody response against viral capsid proteins can be somewhat reduced by encapsulating adenovirus into γ-PGA-CH NPs, as only 3.1% of the encapsulated adenovirus was detected by anti-adenovirus antibodies in the presented work compared to naked adenoviruses. The results obtained and the unique characteristics of the polymer established in this research could provide a reference for the coating and controlled release of viral vectors used in anticancer therapy.

Glutamylation, introduced by tubulin tyrosine ligase-like (TTLL) enzymes, is the most abundant modification of brain tubulin. Essential effector proteins read the tubulin glutamylation pattern, and its misregulation causes neurodegeneration. TTLL glutamylases post-translationally add glutamates to internal glutamates in tubulin carboxy-terminal tails (branch initiation, through an isopeptide bond), and additional glutamates can extend these (elongation). TTLLs are thought to specialize in initiation or elongation, but the mechanistic basis for regioselectivity is unknown. We present cocrystal structures of murine TTLL6 bound to tetrahedral intermediate analogs that delineate key active-site residues that make this enzyme an elongase. We show that TTLL4 is exclusively an initiase and, through combined structural and phylogenetic analyses, engineer TTLL6 into a branch-initiating enzyme. TTLL glycylases add glycines post-translationally to internal glutamates, and we find that the same active-site residues discriminate between initiase and elongase glycylases. These active-site specializations of TTLL glutamylases and glycylases ultimately yield the chemical complexity of cellular microtubules.A combination of structural and protein-chemistry approaches along with phylogenetic analyses provide insights into the specific activities of mouse tubulin tyrosine ligase-like enzymes as initiases or elongases of glutamylation.

Non-spore-forming bacteria can enhance crop salinity tolerance via various strategies, but poor viability during storage and field application limits their use. Inspired by the robust structure of spore dormancy, a core-shell microcapsule consisted of sodium alginate, poly (γ-glutamic acid), and chitosan (APC) is proposed. Using Pantoea alhagi NX-11 as a model, we found that APC encapsulation significantly enhanced bacterial survival during room-temperature storage compared to free cells or alginate beads. This protective effect was further confirmed using two other non-spore-forming strains. The survival mechanism was mainly attributed to the APC-induced metabolic dormancy via suppression of the TCA cycle and oxidative phosphorylation pathways. Furthermore, inoculation with APC-encapsulated NX-11 increased dry weight of rice plant by 24.2% under salt stress in greenhouses and increased grain yield by 15.8% in saline fields, attributing to the enhanced root colonization of NX-11. Overall, this bio-inspired encapsulation strategy provides an effective approach for developing robust microbial inoculants to improve crop resilience in saline soils. Poor storage stability and soil survival limits agricultural application of non-spore forming bacteria. Here, the authors develop a hydrogel microcapsule which improves storage and soil survival, demonstrating application in improving plant salt tolerance under field conditions.