FEBS Letters最新文献

The plasma membrane proton pump is the primary energy transducing, electrogenic ion pump of the plasma membrane in plants and fungi. Compared to its fungal counterpart, the plant plasma membrane proton pump's regulatory C-terminal domain (CTD) contains an additional regulatory segment that links multiple sensory pathways regulating plant cell length through phosphorylation and recruitment of regulatory 14-3-3 proteins. However, a complete structural model of a plant proton pump is lacking. Here, we performed covalent labeling with mass spectrometric analysis (CL-MS) on the Arabidopsis pump AHA2 to identify potential interactions between the CTD and the catalytic domains. Our results suggest that autoinhibition in the plant enzyme is much more structurally complex than in the fungal enzyme.

Dysregulation of human DNA polymerase delta (Polδ) subunits is associated with genome instability and pathological disorders. Genome databases suggest the expression of several spliced variants of subunits which may alter Polδ function. Here, we analyzed the protein-encoding variants of the Polδ subunit p12 and their association with cancer. p12 isoform-2 (p12*) encodes a 79 aa protein with a C-terminal tail distinct from the previously characterized p12. Like p12, p12* dimerizes and interacts with p125 and p50 subunits and is thus an integral component of Polδ. Further, we observed dysregulated p12* expression in low-grade glioma, renal, thyroid, and pancreatic carcinomas. This study identifies a previously unrecognized Polδ complex and highlights a possible regulatory role of p12 variants in cellular phenotypes.

In this research letter, we report the development and validation of a new subset of fluorescence-based CRISPR interference (CRISPRi) tools for our scientific community. The pJL series is directly derived from the original pIRL CRISPRi vectors and conserves all the elements to perform inducible targeted gene repression. These vectors carry two distinct fluorescent markers under the constitutive promoter psmyc to simplify the selection of recombinant clones. We demonstrate the functionality of these vectors by targeting the expression of the glycopeptidolipid translocase mmpL4b and the essential genes rpoB and mmpL3. Finally, we describe an efficient single-step procedure to co-transform mycobacterial species with this integrative genetic tool alongside episomal vectors. Such tools and approaches should be useful to foster discovery in mycobacterial research.

Serratia sp. ATCC 39006 has two tandemly positioned genes, ser4 and ser5, both annotated as sugar aminotransferases, in a putative secondary metabolite biosynthetic gene cluster. Ser5 possesses a complete fold-type I aminotransferase fold, while Ser4 lacks the N- and C-terminal regions and a catalytically important lysine residue of fold-type I aminotransferase. We herein revealed that Ser4 and Ser5 formed a heterotetrameric complex (SerTA) with aminotransferase activity and determined the crystal structures. MD simulations and activity assays with SerTA variants indicated that residues from helix α-8* of inactive Ser4 are important for activity, confirming the importance of heterocomplex formation for activity. Furthermore, the structures suggest that SerTA recognizes a substrate loaded on the carrier protein.

The Federation of European Biochemical Societies (FEBS) was founded in 1964 to bring together the scientific societies of Europe and neighbouring regions and to provide a platform for scientific exchange. Today, FEBS is an organisation of more than 30 000 members across 39 biochemistry and molecular biology societies that promotes excellence in the life sciences in Europe and beyond. To mark the 60^th anniversary of FEBS, FEBS Letters celebrated with a writing contest focussed on participants' visions of scientific societies in the year 2084. Here, we present the winning essay, in which Yussuf Ali (Jagiellonian University, Poland) considers a future in which FEBS transcends not only geographical boundaries, but also interdisciplinary, technological and financial boundaries.

Nanodiscs, consisting of a lipid bilayer surrounded by membrane scaffold proteins (MSPs), are extensively used to study membrane proteins (MPs) because they provide a stable lipid environment. However, the precise mechanism governing MP behavior within the nanodisc remains elusive. Here, we examined the cryo-EM structures of various MPs reconstituted in nanodiscs from EMPIAR. By analyzing the heterogeneity and interactions in the nanodiscs, we discovered that MPs display a distinct spatial preference toward the edges of the nanodisc shells. Furthermore, MPs can establish direct, amphipathic interactions with the MSPs, causing a reduction in local protein dynamics. These interactions may rearrange MSP-MSP interactions into MP-MSP interactions. Collectively, we provide structural insights into how nanodiscs contribute to MP structural behavior and dynamics. Impact statement Nanodiscs are used to study membrane proteins (MPs), but the mechanisms governing the behavior of MPs within nanodiscs remain elusive. Here, we provide structural insights into how nanodiscs contribute to the behavior of MPs, which will aid the interpretation of cryo-EM studies performed using nanodiscs.

Nanobodies (NB) are powerful tools for biotechnological and therapeutic applications. They strongly bind to their targets and are very stable. Early studies showed that NB unfolding is reversible and can be analyzed by equilibrium thermodynamics, whereas more recent studies focused on their kinetic stability in very harsh conditions that are far from storage or physiological temperatures (4-37 °C). Here, we show that the thermodynamic view of NB stability holds in a wide range of temperatures (18-100 °C). The thermodynamic stability of three different NBs did not correlate with binding affinity for their target. Alpha-Fold 2 analyses of these NBs showed structural differences in the binding site and hydrogen bond networks. We expect that our approach will be helpful to improve our capacity to enhance structure-function-stability relationships of NB.

DNA replication and RNA transcription processes compete for the same DNA template and, thus, frequently collide. These transcription–replication collisions are thought to lead to genomic instability, which places a selective pressure on organisms to avoid them. Here, we review the predisposing causes, molecular mechanisms, and downstream consequences of transcription–replication collisions (TRCs) with a strong emphasis on prokaryotic model systems, before contrasting prokaryotic findings with cases in eukaryotic systems. Current research points to genomic structure as the primary determinant of steady-state TRC levels and RNA polymerase regulation as the primary inducer of excess TRCs. We review the proposed mechanisms of TRC-induced DNA damage, attempting to clarify their mechanistic requirements. Finally, we discuss what drives genomes to select against TRCs.

Autophagy is a genetically regulated, eukaryotic catabolic pathway that responds to internal and external cellular signals. In plants, it plays crucial roles in development, and responses to abiotic and biotic stresses. Due to its role in limiting the hypersensitive response, research on the molecular mechanisms of autophagic signalling pathways in plant-microbe interactions has primarily focused on plant-pathogen responses. Although there is substantially less information on the role of autophagy signalling in symbiotic plant-microbe interactions, there is accumulating evidence that it is also a key regulator of mutualistic plant-microbe interactions. Here, we review recent progress on the roles of autophagy in symbiotic plant interactions and discuss potential future research directions. Once understood, the central role that autophagy plays within pathogenic and symbiotic plant-microbe interactions has significant potential application for crop improvement. Manipulating autophagy in legume crops could help support crop growth with reduced levels of fertiliser application while maintaining yields with increased protein content in the harvest.