The FEBS journal最新文献

Isomaltosaccharides, composed of α-(1 → 6)-d-glucosyl residues, exhibit diverse beneficial properties depending on the degree of polymerization (DP) and attract great interest across multiple industries. The anomer-retaining transglucosidase, dextran dextrinase (DDase)-which synthesizes the α-(1 → 6)-d-glucose polymer dextran from α-(1 → 4)-glucan maltooligosaccharides (MOSs)-shows structural similarity to anomer-inverting α-glucohydrolases belonging to glycoside hydrolase family 15 (GH15). Here, we show a new GH15 transglucosidase, α-glucan 4(6)-α-glucosyltransferase, from Tepidibacillus decaturensis (Td46GT) and its mechanism for converting MOS into isomaltooligosaccharide (IMO). Td46GT catalyzes DDase-like d-glucosyl-transfer reactions: α-(1 → 4)-transglucosylation to MOS and α-(1 → 6)-transglucosylation to short-chain MOS of DP 2-3 and IMO. Unlike DDase, it does not produce dextran. Kinetic analyses of the two-substrate reactions revealed that the acceptors determined formed linkages. Relative acceptor-substrate specificity constants (RASCs) indicated that maltotriose and maltose were the best acceptors for α-(1 → 4)- and α-(1 → 6)-transglucosylations, respectively. The subsite affinities calculated from the RASCs were consistent with those obtained from k_cat/K_m values for single-substrate reactions. Time-dependent changes in the MOS and IMO concentrations during the reaction were quantitatively simulated using RASCs and k_cat/K_m values. Structure prediction suggested Td46GT possesses a substrate-binding site similar to DDase, and site-directed mutagenesis identified Phe418 and Tyr612 as critical residues at subsite +2 for MOS and IMO binding. Our results suggested that Td46GT possesses distinct MOS- and IMO-binding subsites in a single active pocket, which are shared by both acceptor and donor substrates, and that the binding manner of the acceptor determines the product specificity of transglucosylation.

Anti-CRISPR (Acr) proteins are small protein inhibitors that block the RNA-guided nucleic acid (DNA or RNA) targeting activity of CRISPR-Cas enzymes. Despite their shared function, Acr proteins display minimal sequence or structural similarity and employ diverse mechanisms to block nuclease activity. Lee and Park characterized the previously undescribed AcrIIA13b protein, which inhibits Cas9 protein. Structural, biochemical, and mutational analyses revealed that AcrIIA13b acts as a DNA mimic, thereby disabling the Cas9 complex from binding to the DNA target.

Progressive aggregation of α-synuclein (α-Syn) in the midbrain, hypothalamus and thalamus is linked to Parkinson's disease (PD), one of the fastest growing neurodegenerative diseases in the U.S. Studies of families with PD history revealed several mutations that are responsible for the early-onset (A30P, E46K, A53T) and late-onset (H50Q) forms of PD. A growing body of evidence indicates that phospho-/sphingolipids and cholesterol alter the aggregation properties of wild-type (WT) α-syn. However, the effects of these lipids on the rate of α-syn mutants remain unclear. In the current study, we determined the aggregation rates of A30P, E46K, A53T, H50Q and WT α-syn in the presence of large unilamellar vesicles composed of phosphatidylcholine (PC), sphingomyelin (SM) and cholesterol (Cho)-the key lipids of neuronal membranes. We also utilised a set of biophysical methods to reveal the extent to which lipids alter the morphology and secondary structure of amyloid fibrils. We found that familial mutations uniquely altered α-syn interactions with lipid bilayers, which resulted in the altered rate of protein aggregation in the presence of lipid bilayers. Furthermore, A30P mutation fully disabled α-syn interaction with LUVs, while E46K, A53T and H50Q mutations altered cytotoxicity of α-syn fibrils formed in the presence of lipid bilayers. These results suggest that changes in plasma membrane lipid profiles may have a strong effect on the onset and progression of PD in individuals with familial mutations.

Although macrophages (MФs) are vital regulators of acute kidney injury (AKI), their diverse roles in renal injury and repair remain elusive. Li et al. leveraged single-cell RNA sequencing to dissect MФ dynamics at different stages of cisplatin-induced AKI. They identified four distinct renal MФ subsets, in which monocyte-derived MФs (Mo-MФs) drive major renal inflammation during AKI progression, whereas renal resident Cx3cr1⁺ MФs promote renal repair via AXL-GAS6 signaling-mediated efferocytosis during AKI regression. This work advances our understanding of innate immune responses in AKI and provides insights into the heterogeneity of MФs in renal injury and repair.

We highlight original articles published in The FEBS Journal in 2023 and 2024 that members of our Editorial Board deemed particularly notable. The papers discussed here received nominations based on their scientific excellence, timeliness and broad appeal. These outstanding original articles span a broad range of topics related to the molecular life sciences. We invite you to revisit these gems and let us know your favourites.

Porcine reproductive and respiratory syndrome virus (PRRSV) is a highly infectious RNA virus that severely affects swine herds, causing respiratory and reproductive disorders with major global economic impact. Current first-generation vaccines based on inactivated or attenuated viruses offer limited protection and may pose safety risks. Given the virus's high mutation rate and genetic variability, there is a growing need for safer and more effective vaccines. Plant molecular farming represents a promising alternative production system for second-generation subunit vaccines, offering advantages in terms of cost, biosafety, and scalability. Recent studies have explored the coupling of PRRSV antigens to virus-like (VLP) or nonviral self-assembling protein nanoparticles (PNPs) to enhance immunogenicity. VanderBurgt et al. now provide a successful example of the production of both VLP and PNP vaccines against PRRSV in plants, with preliminary in vivo results showing encouraging immune responses.

Microorganisms living in cold environments such as the Antarctic and deep sea usually possess cold-adapted enzymes, which are known to have high catalytic efficiency and low stability owing to their flexible structures. Research on cold-adapted enzymes has not progressed much due to the challenge of these enzymes being less stable. However, several cold-adapted enzymes with high thermal stability have recently been reported. In this study, we investigated the biochemical properties of glucokinases from the psychrophilic Pseudoalteromonas sp. AS-131 (PsGK) isolated from the Antarctic Ocean and mesophilic Escherichia coli (EcGK). We demonstrated that PsGK is a cold-adapted enzyme with high thermal stability. A comparison of the crystal structures and spectroscopic studies revealed that PsGK has an additional disulfide bond connecting the N and C termini. To test whether this bond is important for stability, we prepared a PsGK variant by removing the bond and observed the significant reduction in thermal stability. In addition, the introduction of the artificial disulfide bonds in homologous positions in EcGK increased the thermal stability without the reduction in maximum activity. These results confirmed that the introduction of a disulfide bond at the proper position, such as the connection of the N and C termini, significantly improved stability without changing the nature of enzymes. Our findings propose a new strategy that will contribute to the industrial application of enzymes.

Protein kinase A (PKA) regulatory subunit Iβ (RIβ) plays a crucial role in modulating PKA activity through its interaction with the catalytic (C) subunit. Recent studies have identified two variants of RIβ that have not been distinguished until now. The variants differ in a single residue at Position 268 (alanine vs arginine), located within one of two structural motifs known as N3A motifs. Our study reveals distinct biochemical functions of the variants, highlighting the role of the second N3A motif at the N terminus of the cyclic nucleotide binding domain B (CNB-B). We demonstrate an enhanced binding affinity of the A268 variant for cAMP and an altered interaction with the C-subunit. Substitution in the N3A^B motif also affects the cooperativity between the cAMP binding sites as well as kinase activity in the absence of cAMP. In HEK293 cells, we demonstrated a reduced cAMP-induced dissociation of RIβ R268 PKA holoenzymes in a time-dependent manner. Gaussian molecular dynamics simulations revealed that the CNB-A domain is more flexible in A268 while the opposite is true for the CNB-B domain, which is more dynamic in the R268 protein. This study underscores the importance of distinguishing between the two RIβ variants, as they exhibit distinct biochemical properties that alter PKA regulation. Comparison of cellular localization showed that both variants form small droplets in the cytoplasm that colocalize with the C-subunit in the presence of cAMP. These findings suggest that both RIβ variants undergo liquid-liquid phase separation, similar to RIα.

Cotranslational subunit assembly is thought to be a prominent feature throughout the proteome, but, in bacteria, there are only a limited number of experimentally confirmed examples and most involve the addition of extraneous tag sequences for experimental convenience. Toxin CcdA and Antitoxin CcdB are components of the ccdAB operon. They assemble in a hetero-multimeric complex in vivo. Building on a previously characterised saturation mutagenesis dataset of CcdB, we investigated how operonic gene organisation influences the cotranslational folding and assembly of the toxin-antitoxin complex. We compared the phenotypic effects of CcdB mutations when expressed alone versus in the native operonic context downstream of CcdA. Although several charged and polar mutations in the CcdB core result in loss of function in the absence of CcdA, many of these are functionally rescued in the operonic context. Furthermore, we show that the efficiency of rescue is substantially reduced when ccdA and ccdB are expressed from separate mRNAs rather than from a single polycistronic transcript. Our results highlight a direct role for cotranslational interactions in enabling correct folding of CcdB and suggest that bacterial operon structure may have evolved, in part, to facilitate such chaperone-like rescue of unstable protein variants. Gene organisation in operons in bacteria may thus reflect a fundamental cotranslational mechanism that is important for the effective assembly of protein complexes and can potentially buffer substantial genetic variation.

Mucopolysaccharidosis type II, also known as Hunter syndrome, is a rare and fatal disease caused by mutations in the iduronate 2-sulfatase (IDS) encoding gene. Enzymatically inactive IDS variants lead to pathological accumulation of glycosaminoglycans in lysosomes, resulting in dysfunction of multiple organs. IDS is expressed as a precursor protein, and its proper processing and lysosomal targeting are crucial for enzymatic activity. However, the intracellular dynamics of IDS remain poorly understood, and a better understanding of its processing mechanisms would benefit the development of new therapeutic strategies. alphafold 3 predicted an interaction between IDS and the E3 ubiquitin ligase RNF13. Co-immunoprecipitation assays confirmed this interaction and further revealed that RNF13 preferentially interacts with a predominantly underglycosylated immature form of IDS, leading to altered IDS glycosylation and maturation. The results demonstrate that IDS glycosylation site Asn246 is important for lysosomal targeting, although its glycosylation is not altered by RNF13. Importantly, this study demonstrates that RNF13 forms a heterodimer with the E3 ubiquitin ligase RNF167, which modulates the lysosomal trafficking of both proteins. In addition, the heterodimer interacts and alters IDS processing differently than RNF13 or RNF167 alone. RNF13 catalytic E3 ligase activity is required to generate an underglycosylated form, but not that of RNF167. This study shows that the proteasome rapidly degrades IDS underglycosylated forms, and RNF13 exerts a protective effect. Overall, this study reveals a previously undescribed and dual role of RNF13 in IDS maturation and degradation, providing mechanistic insights into IDS trafficking.