FEBS Letters最新文献

Multiple system atrophy (MSA) is a progressive neurodegenerative disease characterized by accumulation of α-synuclein cross-β amyloid filaments in the brain. Previous structural studies of these filaments by cryo-electron microscopy (cryo-EM) revealed three discrete folds distinct from α-synuclein filaments associated with other neurodegenerative diseases. Here, we use cryo-EM to identify a novel, low-populated MSA filament subtype (designated Type I₂) in addition to a predominant class comprising MSA Type II₂ filaments. The 3.3-Å resolution structure of the Type I₂ filament reveals a fold consisting of two asymmetric protofilaments, one of which adopts a novel structure that is chimeric between two previously reported protofilaments. These results further define MSA-specific folds of α-synuclein filaments and have implications for designing MSA diagnostics and therapeutics.

Cancer-associated fibroblasts (CAFs) are the dominant nonmalignant component of the tumour microenvironment (TME). CAFs demonstrate a high level of inter- and intra-tumour heterogeneity in solid tumours, though the drivers of CAF subpopulations are not fully understood. Here, we demonstrate that non-small cell lung cancer (NSCLC) patient-derived CAFs upregulate the secretion of inflammatory cytokines (IL6, LIF, IL33, GM-CSF, IL1ra) and chemokines (CCL2, CCL3, CCL4, CCL20, CXCL8, CXCL9, CXCL10, CXCL11) in response to in vitro co-culture with anti-CD3/anti-CD28-stimulated peripheral blood mononuclear cells (PBMCs) via IFNγ and TNFα. Furthermore, T-cell-derived IFNγ inhibits CXCL12 secretion by CAFs in vitro. Our results highlight the ability of T-cell effector cytokines to modulate the CAF secretome in NSCLC.

Some tumors employ a mechanism called alternative lengthening of telomeres (ALT) to counteract telomere shortening-induced replicative senescence. Several hallmarks are used to identify cell lines and tumors as ALT-positive. Here, we analyzed a panel of ALT-positive and -negative cancer cell lines to investigate the specificity and sensibility of ALT-associated markers. We found that all the markers showed high sensitivity, indicating that cells not showing ALT markers are not ALT cells. Conversely, specificity varied significantly, i.e., many markers yield false positives. Detection of false positives may have influenced previous estimations of ALT incidence among tumors. Moreover, claims on the 'coexistence' of ALT and telomerase perhaps should be reconsidered. The findings prompt further study into the nature of these markers and their roles as either part of the ALT machinery or as by-products.

Detecting circulating tumor cells (CTCs) is challenging due to their low presence and heterogeneity. Traditional methods using EpCAM-based separation struggle with CTCs that have undergone epithelial-mesenchymal transition, as this results in lower EpCAM expression. This study presents the use of silica-coated magnetic nanobeads functionalized with streptavidin for CTC capture. Using the FETCH magnetic separation system, we validated the capture efficiency of our beads on tumor cells with varying EpCAM expression. Our beads showed superior capture rates for LNCaP (97%), PC3-9 (91%), PC3 (23%), A549 (22%), and T24 (8%) cells compared to commercial MojoSort™ beads. Despite slightly higher nonspecific binding than CellSearch, our beads demonstrated improved sensitivity for EpCAMlow cells, suggesting they have promise for enhanced CTC capture.

PIWI-interacting RNAs (piRNAs) were discovered in the early 2000s and became known for their role in protecting the germline genome against mobile genetic elements. Successively, piRNAs were also detected in the somatic cells of gonads in multiple animal species. In recent years, piRNAs have been reported in non-gonadal tissues in various arthropods, contrary to the initial assumptions of piRNAs being exclusive to gonads. Here, we performed an extensive literature review, which revealed that reports on non-gonadal somatic piRNA expression are not limited to a few specific species. Instead, when multiple studies are considered collectively, it appears to be a widespread phenomenon across arthropods. Furthermore, we systematically analyzed 168 publicly available small RNA-seq datasets from diverse tissues in 17 species, which further supported the bibliographic reports that piRNAs are expressed across tissues and species in Arthropoda.

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 Homo sapiens Na⁺/H⁺ antiporter NHA2 (SLC9B2) transports Na⁺ or Li⁺ in exchange for protons across cell membranes, and its dysfunction results in various pathologies. The activity of HsNHA2 is specifically inhibited by the flavonoid phloretin. Using bioinformatic modeling, we predicted two amino acids (R177 and S178) as being important for the binding of phloretin to the HsNHA2 molecule. Functional expression of HsNHA2 in Saccharomyces cerevisiae and its site-directed mutagenesis revealed that while the R177T mutation resulted in an antiporter that was less sensitive to phloretin, the S178T mutation enhanced the inhibitory effect of phloretin on HsNHA2. Our data corroborate the transport properties of HsNHA2 and its interactions with an inhibitor and can be helpful for the development of new therapeutics targeting this antiporter and its pleiotropic physiological functions.

Fluorescence resonance energy transfer (FRET)-based biosensors are powerful tools for studying second messengers with high temporal and spatial resolution. FRET is commonly detected by ratio imaging, but fluorescence lifetime imaging microscopy (FLIM), which measures the donor fluorophore's lifetime, offers a robust and more quantitative alternative. We have introduced and optimized four generations of FRET sensors for cAMP, based on the effector molecule Epac1, including variants for either ratio imaging or FLIM detection. Recently, Massengill and colleagues introduced additional mutations that improve cytosolic localization in these sensors, focusing on constructs optimized for ratio imaging. Here we present and briefly characterize these mutations in our dedicated FLIM sensors, finding they enhance cytosolic localization while maintaining performance comparable to original constructs.

CRISPR-Cas9 is a widely used genome-editing tool. We previously developed a method with improved homology-directed repair efficiency and reduced off-target effects by utilizing a fusion protein of AcrIIA4, a Cas9 inhibitor, and Cdt1, which accumulates in the G1 phase and activates Cas9 only in the S/G2 phase. However, it is unknown whether Cas9 inhibition by AcrIIA4 + Cdt1 occurs repeatedly in the G1 phase as the cell cycle progresses. In this study, we used the CRISPRa system to monitor changes in the interaction between Cas9 and AcrIIA4 + Cdt1 at single-cell resolution and in real time. Our findings are among the few examples of successful detection of fluctuating protein-protein interactions that oscillate over time.

Hermansky-Pudlak syndrome type 1 (HPS-1) is a rare, autosomal recessive disorder caused by defects in the biogenesis of lysosome-related organelles complex-3 (BLOC-3). Impaired kidney function is among its clinical manifestations. To investigate HPS-1 renal involvement, we employed 1D-gel-LC-MS/MS and compared the protein composition of urinary extracellular vesicles (uEVs) from HPS-1 patients to normal control individuals. We identified 1029 proteins, 149 of which were altered in HPS-1 uEVs. Ingenuity Pathway Analysis revealed disruptions in mitochondrial function and the LXR/RXR pathway that regulates lipid metabolism, which is supported by our novel Hps1 knockout mouse. Serum concentration of the LXR/RXR pathway protein ApoA1 in our patient cohort was positively correlated with kidney function (with the estimated glomerular filtration rate or eGFR). uEVs can be used to study epithelial cell protein trafficking in HPS-1 and may provide outcome measures for HPS-1 therapeutic interventions.