FEBS Letters

Multiple peptide resistance factor (MprF) are bi-functional enzymes encoded by several bacterial species and carry out the transfer of an amino acid from a charged tRNA to the lipid head group and further translocate the lipid across the membrane. Biochemical studies have revealed that the soluble synthase domain generates specificity and the structures of MprF have defined the general architecture of these enzymes, and that they can exist in different oligomeric states. Here, we characterise the gene product of lpiA, a MprF homologue from Agrobacterium fabrum (formerly called A. tumefaciens strain C58), a microbe that is commonly used in plant molecular biology. Cryo-EM analysis of AfMprF reveals a dimeric structure both in detergent micelle and in lipid nanodisc, and similar in architecture to the homologous enzyme from related Rhizobium sp. We further analyse some conserved residues in the soluble domain and suggest that the sulphur-aromatic motifs play a key role in substrate binding. Similar architecture of enzymes in closely related bacterial species of Agrobacterium and Rhizobium hints an evolutionary relationship but the importance of these oligomeric states in vivo remains to be analysed.

Conflicting roles have been proposed for the E. coli protein RatA. Initially described as a ribosome targeting toxin, a later report pronounced it the bacterial homologue to the inner mitochondrial membrane protein Coq10. Coq10 proteins are conserved from prokaryotes to human and implicated to serve a lipid chaperone role in the biosynthesis of ubiquinone, a crucial electron carrier during aerobic respiration. We recently identified that the contradictory results published for RatA can be attributed to a mis-annotation of the gene in the reference genome. Here, we further elucidate the molecular function of RatA. We clarify that RatA is not a toxin but serves as a lipid shuttle for ubiquinone from its cytosolic biosynthesis complex to the inner membrane. Furthermore, we show that the loss of RatA results in an impaired, but not abolished electron transport chain and demonstrate broad metabolic adaptations of the cells as a consequence. Therefore, we propose to rename RatA to UbiM to reflect its function and to be in accordance with the naming convention of other ubiquinone biosynthesis proteins.

Rhodococcus opacus is an oleaginous actinobacterium with considerable potential for lipid-based bioproduction, as well as for utilising a variety of carbon sources as substrates, including renewable, cost-effective resources. Although its capacity for triacylglycerol accumulation is well established, the regulatory logic that governs its fatty acid and mycolic acid biosynthesis is still poorly understood. Here, we investigated the transcriptional control of the type II fatty acid synthase (FASII) pathway in R. opacus PD630, revealing a regulatory architecture that is more complex than previously assumed. Differential gene expression analysis showed that environmental cues, including temperature, pH, carbon-to-nitrogen ratio and the presence of free fatty acids influence the FASII gene cluster expression in a non-uniform manner. This phenomenon suggests the presence of internal transcription start sites and modular regulation within the cluster. We identified three lipid-responsive transcription factors, MabRRO, FadR1RO and FadR2RO, that are all capable of binding the fasII promoter in vitro. DNA binding of FadR1RO and FadR2RO was disrupted by long-chain acyl-CoA molecules, indicating ligand-dependent control. Together, these findings reveal previously unrecognised layers of transcriptional regulation in the R. opacus FASII pathway and highlight both conserved and divergent regulatory features within the Mycobacteriales lineage. Featured Image O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=134 SRC="FIGDIR/small/678588v2_ufig1.gif" ALT="Figure 1"> View larger version (22K): org.highwire.dtl.DTLVardef@456a6dorg.highwire.dtl.DTLVardef@14e3d1forg.highwire.dtl.DTLVardef@18ed368org.highwire.dtl.DTLVardef@1d979a6_HPS_FORMAT_FIGEXP M_FIG C_FIG

Transcriptional termination efficiency is considered an important parameter for fine tuning bacterial gene expression. Still, the design principles that determine transcription termination efficiency remain poorly understood. In this study, we aimed to investigate the impact of the 3' untranslated region (3'UTR) on gene expression in Escherichia coli and other bacteria. First, 3'UTR variant sequences were generated, with randomized 30 bp sequences inserted between the STOP-codon and an intrinsic terminator, consisting of a GC-rich hairpin and a downstream poly(U)-tail. Using three reporter genes, it was found that different 3'UTR sequences resulted in an up to five-fold difference in protein production, independent of the upstream coding sequence. The highest protein production was achieved when an adenosine was present directly upstream of the terminator hairpin. This was consolidated by systematic substitution of key nucleotides of the terminator and assessing their effect on mRNA and protein levels. Subsequently, we developed a predictive random forest machine learning model trained on the termination efficiency of different natural and synthetic terminator sequences, revealing an important role for the nucleotides directly upstream of the terminator hairpin. Altogether, this study showed that an additional adenosine nucleotide upstream of the terminator hairpin leads to improved protein production while reducing terminator read-through.

The human mitochondrial amidoxime reducing component 1 (mARC1) is a molybdenum-dependent enzyme whose protein-coding variants confer protection against common metabolic liver diseases. Whereas the frequent A165T variant acts largely through accelerated cellular degradation, the basis of protection by the rarer M187K variant remains obscure, as it has been reported that there are no differences between the "wild-type" and M187K variant protein in terms of cellular protein levels and localisation. Here, the crystal structure of the human mARC1 M187K variant, crystallised as a T4 lysozyme fusion after iterative micro-seeding, was determined at 1.63 [A] resolution. The variant structure is essentially superimposable with the previously reported "wild-type" and A165T structures, with pairwise root-mean-square deviations of 0.3-0.4 [A], and the pentacoordinated molybdenum cofactor is fully intact. Differential scanning fluorimetry across a broad pH range revealed only a modest, pH-dependent decrease in thermal stability associated with the exchange, most pronounced at alkaline pH. These data suggest that the disease-protective effect of M187K is unlikely to originate from gross structural rearrangement or active-site perturbation. SynopsisThe structure of the disease-relevant human mARC1 M187K variant was determined at 1.63 [A] resolution after iterative micro-seeding. The variant does not display relevant perturbations of the overall protein fold or active site structure, but differential scanning fluorimetry detects a pH-dependent decrease in overall protein stability associated with the M187K amino acid exchange.

Mitochondrial potassium channels play an important role in regulating cellular metabolism, redox balance, and survival, particularly in excitable tissues such as the heart. Among them, the mitochondrial large-conductance calcium-activated potassium (mitoBKCa) channel has been implicated in cardioprotection during ischemia-reperfusion injury. At the same time, growing evidence indicates that mitochondria act as light responsive organelles, with cytochrome c oxidase (COX) serving as a primary chromophore for red and near-infrared (NIR) light. In this study, we investigated whether 820 nm infrared light modulates mitoBKCa channel activity in mitochondria isolated from guinea pig cardiomyocytes. Using patch-clamp recordings of mitoplasts, we demonstrated that illumination at 820 nm NIR wavelength enhanced mitoBKCa channel activity in a redox-dependent manner. Our findings reveal a previously unrecognized mechanism linking NIR light modulation via COX to the regulation of cardiac mitoBKCa channels as a metabolic sensor. This study identifies the mitoBKCa channel as a novel effector of light-induced mitochondrial signaling and suggests that modulation of cardiac mitochondrial potassium transport by NIR light may contribute to cardioprotective effects. These results provide new insight into the integration of bioenergetic and photoregulatory processes in mitochondria and support the development of non-pharmacological strategies targeting mitochondrial function.

Glycine substitutions in the collagen triple helix cause diverse heritable disorders, but their effects vary with local sequence environment. We tested whether sequence context helps determine the consequences of Gly replacement by combining case-weighted bioinformatic analysis, thermodynamic measurements on collagen model peptides (CMPs), and all-atom molecular dynamics simulations. Case-weighted analysis of pathogenic COL3A1 glycine substitutions identified Pro immediately following the substituted Gly, corresponding to a GP context, as the strongest enriched local feature. To examine this experimentally, we designed CMPs with stabilizing terminal segments flanking native collagen sequence windows containing clinically observed Gly[->]Ser and Gly[->]Arg substitutions. Gly[->]Arg substitutions were generally more destabilizing than Gly[->]Ser at the same site. However, the strongest effect was sequence-dependent: within the Gly[->]Ser class, GP-site substitutions caused larger losses of thermal stability and unfolding enthalpy than nonGP substitutions, and some GP-site Gly[->]Ser mutations were as destabilizing as Gly[->]Arg substitutions. Molecular dynamics simulations showed that all peptides remained globally triple-helical, but GP-site mutants exhibited greater loss of canonical interchain hydrogen bonds and reduced local backbone accommodation. Thus, the effect of glycine substitution in collagen depends not only on the replacing residue but also on the permissiveness of the surrounding sequence, with Pro-adjacent sites representing especially restrictive local environments. Statement of SignificanceCollagen diseases often result from replacement of a required glycine in the triple helix, but the same substitution can have very different consequences at different sites. Using clinical bioinformatics, collagen model peptides, and molecular dynamics simulations, we show that the surrounding sequence is a major determinant of mutational outcome. The most restrictive context identified here is a GP site, in which proline immediately follows the substituted glycine: Gly[->]Ser mutations at GP sites are more destabilizing than Gly[->]Ser mutations at sites without a following proline and can be as damaging as Gly[->]Arg substitutions. These results define a sequence-based rule for collagen mutational tolerance relevant to both triple-helix stability and interpretation of pathogenic variants.

Mechanotransduction via the actin cytoskeleton is linked to fundamental cellular processes such as morphogenesis, cell division, and motility, requiring the control of tensile forces mediated by the motor protein non-muscle myosin 2 (NM2). Formins such as mDia1 have been shown to elongate actin structures that are under mechanical tension; conversely, mDia1s elongation rates are modulated by the applied force. Despite their relevance at the membrane/cortex interface, reported values for tension in formin-elongated actin filaments stem from theoretical estimates and simulations, but have not been amenable experimentally so far. Thus, we developed a Forster resonance energy transfer (FRET)-based, tension-sensitive probe (mDia1TS) and quantified the measured tension in live U2OS cells using fluorescence lifetime imaging microscopy (FLIM). Through whole-cell ROI analysis we show a short and long lifetime component, reporting an intensity-weighted, averaged lifetime corresponding to [~]3.5 pN. Upon mitogen stimulation of cells using EGF, we show that the tension homeostasis changed significantly, with a measurable increase in tension in the cells periphery and relaxation in its center. Furthermore, the reported average tension relaxed by 2 pN after adding the NM2 inhibitor para-nitroblebbistatin. We utilized siRNA knockdowns of individual NM2 paralogs (NM2-A, NM2-B, or NM2-C) to measure their individual contribution, revealing NM2-A as the main paralog to produce tensile force in this system. Taken together, we demonstrate that mDia1TS is able to directly determine that active mDia1 in cells is under tension, and that subcellular quantification with pN precision is possible. SignificanceDespite the fundamental importance of formins in regulating actin-based processes, reported values for tension in formin-mediated actin structures stem from simulations and theoretical estimates. In this study we developed a FRET-based, tension-sensitive reporter probe for formin mDia1, which we termed mDia1TS. Given the expanding clinical spectrum of DIAPH1/mDia1 mutations, our tool mDia1TS provides a quantitative tool for elucidation of changes in cytoskeletal assemblies.

In AL amyloidosis, monoclonal immunoglobulin light chains (LCs) aggregate as amyloid fibrils in tissues. In synergy with the intrinsic aggregation propensity of specific LC sequences, microenvironment factors may be involved in tuning the disease pathophysiology, in particular proteolytic LC remodelling and heterotypic interactions in the extracellular milieu. Accounting for extrinsic modulators is critical for understanding the phenotypic variability of AL, usually imputed mainly to the LC diversity. We investigated the effects of apolipoprotein E (allele 3, apoE3) and clusterin (CLU), two amyloid-signature proteins involved in extracellular proteostasis, on the fibrillogenesis kinetics of amyloidogenic LC fragments from patient-derived sequences, as well as on aggregate composition, fibril morphology and thermodynamic stability. We show that apoE3 and CLU act as heterotypic interactors of prefibrillar and fibrillar LCs, significantly modulating LC amyloidogenesis, with complex and non-monotypic effects that range from anti- to pro-amyloidogenic depending on their concentration and on the LCs intrinsic amyloidogenicity. ApoE3 and CLU also influence fibril morphology, possibly by modifying protofilament association, and alter their thermodynamic properties. LC interactors may play a significant and insofar underappreciated role in the AL pathophysiology in vivo, likely contributing to phenotypic variability and structural polymorphisms and, possibly, to fibril resilience to amyloid reabsorption strategies.

RationaleFibrotic lung diseases, such as idiopathic pulmonary fibrosis (IPF) and fibroproliferative remodeling in acute respiratory distress syndrome (ARDS), are characterized by increased extracellular matrix (ECM) deposition. However, measuring collagen accumulation alone does not capture differences in ECM organization or biochemical maturation that may distinguish persistent fibrosis from potentially reversible remodeling. ObjectivesTo examine collagen organization characteristics and mature (pyridinoline) collagen crosslinking amount in established end stage fibrotic lung disease (IPF) and fibroproliferation following an acutely damaged lung (non-resolving (NR) ARDS) and to investigate any relationships in these parameters and temporal tissue remodeling. MethodsHuman lung tissue samples from control subjects, patients with IPF, and NR-ARDS were analyzed. Collagen amount and fiber organization were digitally quantified using picrosirius red staining. Mature collagen crosslinking was assessed by quantification of pyridinoline crosslinks. Measurements and Main ResultsLung tissue from both IPF and NR-ARDS lungs had higher collagen content compared with controls. Collagen fiber organization differed between groups. IPF lungs exhibited collagen architectures consistent with established fibrosis, whereas NR-ARDS lungs showed altered but less stabilized collagen organization despite similarly elevated collagen levels. Mature collagen crosslinks were significantly higher in IPF lungs but not in NR-ARDS lungs compared to controls. Integrated analyses identified distinct disease-associated ECM phenotypes, indicating that higher collagen abundance in NR-ARDS, unlike IPF, is not accompanied by more mature and persistent collagen crosslinking. ConclusionsDespite shared increases in collagen content, IPF and NR-ARDS lungs differ fundamentally in collagen organization and crosslinking maturity, suggesting differences in the reversibility of these conditions.

IntroductionPressure sores are a major health problem in people with spinal cord injury resulting in ischaemic tissue lesions caused by prolonged pressure against a bony surface. Conventional therapies are often defective and fundamental researches on the healing process of pressure sores must be enriched in order to understand any novel therapies that may be applied. We focalize on pressure sores fibroblasts as dermal fibroblasts perform a critic role in wound healing by populating the wound site to produce extracellular matrix. After characterizing morphological and the genetic profile of healthy fibroblasts and fibroblasts from pressure ulcers, we conducted an analysis of fibroblast proliferation, migration and myofibroblastic differentiation capacity. Materials and Methodsafter acquisition of dermal explants and fibroblasts culture, we conducted histological analysis, an evaluation of gene expression by RT-qPCR and an assessment of fibroblasts proliferation and migration capacity through IncuCyte. A study of the differentiation of fibroblasts into myofibroblasts through the detection of Alpha-Smooth Muscle Actin (-SMA) expression by immunofluorescence was also conducted. Resultshistological analysis showed histological analysis showed dermal disorganization in pressure sore compared with health skin, differences in morphological aspects and density of fibroblasts. Pressure sore fibroblasts express less genes coding for ECM proteins, metalloproteases, collagen III, Connective tissue growth factor (CTGF) and ACTA2 coding for -SMA. Pathological fibroblasts appear to proliferate less quickly than healthy fibroblasts but no differences in migration capacity were found. After stimulation under TGF-{beta}, pressure sore fibroblasts lose their ability to differentiate into myofibroblasts compared to healthy fibroblasts and this could be in relation with a less expression of ACTA2. ConclusionAll of our results highlight a morphological, genetic and functional difference between healthy and pathological fibroblasts which have a modified phenotype, less effective for skin repair. This suggests that new therapies for chronic wounds must take into account the environment in which they are applied and that pathological cells do not necessarily respond to treatments in the same way as healthy cells. Our results are not statistically significant, although several trends emerge. This is explained by the heterogeneity of the patients medical history and requires repetition of the experiments.

Protein homeostasis - i.e., proteostasis - is the biological process by which the qualitative and quantitative balance of the proteome is conducted, either by preserving functionally relevant proteins or by degrading unnecessary ones. Stress conditions can modulate cellular proteostasis in order to promote cytoprotection and preserve the viability of living organisms. Among the cellular pathways already described that can play an important role in preserving biological functions by modulating proteostasis are the heat shock response and the ribosome quality control pathways. In this work, we show that the Rqc1p protein is necessary for the thermoadaptation of S. cerevisiae to heat shock, as RQC1-deficient yeast is sensitive to elevated temperatures. In silico approaches - such as multiple sequence alignment, structural analysis, and molecular dynamics simulations - confirmed earlier predictions that Rqc1p shares characteristics with the bHLH family of proteins. We also verified, through computational prediction of sub-cellular localization, that S. cerevisiae Rqc1p contains nuclear localization signals, suggesting that this protein can potentially be translocated toward the nucleus, thereby broadening its current range of recognized biological functions in this organism. Also, analysis of yeast transcriptomes subjected to heat shock showed that Rqc1p mRNA levels do not fluctuate in response to heat shock, suggesting that cellular concentrations of Rqc1p are already at optimal levels to elicit a rapid and effective response during thermal stress in S. cerevisiae.

Age-related macular degeneration (AMD) is a progressive complex eye disease and one of the leading causes of blindness. AMD progression is marked by molecular changes in the retinal pigmented epithelium (RPE) which include increased reactive oxygen species (ROS) accumulation, mitochondrial dysfunction - eventually leading to dysfunctional RPE. Mitophagy regulator, Pink1, is reduced in the RPE of AMD patients and Pink1 loss leads to a shift from mitochondrial respiration to glycolysis. Serine is a non-essential amino acid which is de novo synthesized from glycolytic intermediate 3-PG via the rate limiting enzyme PHGDH. Serine is tightly integrated into anabolic processes like glutathione (GSH) cycling, maintaining NADH/NADPH pools leading to changes in AMPK signaling. Here, we show that Pink1 loss leads to a reduction in PHGDH and serine levels in the RPE leading to impaired mitochondrial structure and function, increased ROS mediated damage, increased inflammation, and hampered retinal function. Serine supplementation rescued ROS accumulation, balanced GSH abundance, and increased retinal function. Overall, our study highlights the potential of dietary serine in ROS management in AMD.

Eukaryotes use several distinct quality control pathways to resolve aberrant ribosomes and mRNAs. For example, the no-go decay mRNA pathway is stimulated after ribosome collisions caused by stalled ribosomes translating damaged or truncated mRNAs. Separate decay pathways for non-functional 40S and 60S subunits containing rRNA mutations affecting decoding and peptidyl transferase activity, respectively, have also been elucidated. To our knowledge, whether eukaryotes have evolved a quality control pathway to sense and process globally stalled ribosomes is unclear; however, such a pathway would be advantageous to eukaryotes during exposure to natural elongation inhibitors such as ricin and diphtheria toxin. Here, we test how prolonged robust inhibition of elongation using a high dose of cycloheximide (CHX) affects ribosome turnover. Despite no decrease in cell viability and that mammalian ribosomes have been classically characterized of having a half-life of 3-5 days, a single 24 hr high dose of CHX resulted in drastically shortened half-lives (<24 hr) of 28S and 18S rRNA in A549 cells. A [~]2-fold reduction in nearly all ribosome species was observed by polysome analysis in HeLa and A549 cells after prolonged CHX treatment. Depletion of ribosomes was also evident when assessing ribosomal proteins from both the 40S and 60S subunits by Western blot. Literature supports that ribosomes can be degraded by autophagy and the ubiquitin (Ub)-proteasome system. Upon testing inhibitors of both pathways, only proteasome inhibitors (i.e., MG132 and bortezomib) rescued both rRNA and ribosomal protein levels. Proteasome inhibitors also rescued ribosome levels in polysome profiling experiments. Remarkably, rRNA levels were not rescued during CHX treatment when co-treated with the Ub activating enzyme E1 inhibitor, TAK243. Polysome analysis also showed that the high prolonged dose of CHX did not cause robust accumulation of collided ribosomes compared to control treatments. Proteasome-dependent turnover of rRNA was also observed with high doses of other elongation inhibitors, namely anisomycin, homoharringtonine, and lactimidomycin. The recognition capabilities of the pathway were further expanded as we observed that 80S ribosomes not trapped on the mRNA were also targeted for degradation by the proteasome. Together, our findings define the framework of a regulatory pathway in mammalian cells that degrades both ribosomal subunits in response to prolonged periods of robust elongation inhibition.

Microtubules are dynamic filaments of tubulin heterodimers that comprise an essential part of the eukaryotic cytoskeleton1. The nucleotide state of tubulin controls microtubule dynamics: stable GTP-microtubules favor polymerization, whereas unstable GDP-microtubules drive depolymerization2. Anticancer compounds such as Taxol (paclitaxel) target microtubule dynamicity by preventing microtubule depolymerization3,4. Despite decades of work, the molecular basis of microtubule dynamics remains poorly defined. Using cryo-EM, we determined [~]2.2 [A] structures of human microtubules in GTP-like (GMPCPP) and GDP states. Comparison of these two states revealed switch-like structural changes as tubulins transition from the pre-hydrolysis (GMPCPP) to the post-hydrolysis (GDP) state. Additional structure determination of Taxol-bound microtubules at [~]2.2 [A] showed that Taxol binding converts the microtubule lattice into a pre-hydrolysis state by reversing the structural switches flipped during GTP hydrolysis. Focusing our analysis on the microtubule seam shows that the pre-hydrolysis conformation of GMPCPP or Taxol-GDP exhibits favorable lateral interactions at the seam, with lattice deformations clearly visible at the GDP seam. Together, our data show the existence of structural switches in tubulin that are coupled to the nucleotide state and are exploited by Taxol to stabilize microtubules into a pre-hydrolysis-like state. (191 words)

Pulmonary vascular remodelling is common in patients with chronic obstructive pulmonary disease (COPD). Vascular endothelial growth factors (VEGFs) are key mediators in angiogenesis and vascular remodelling and exist in different isoforms. VEGF-A is the most potent angiogenic member binding to VEGF receptor 2 (VEGFR2). There are, however, few studies on other isoforms, as VEGF-C, and its receptor VEGFR3 in COPD and subsequent impact of cAMP therapies on VEGF isoforms. Our aim was to evaluate the VEGF isoform synthesis in primary distal lung fibroblasts from control subjects (non-smokers (n=6) and ex-smokers (n=4), and COPD subjects with GOLD stage II (n=4) or GOLD stage IV (n=6), and the expression of VEGFR2 and VEGFR3 in human lung tissue. Primary lung fibroblasts were exposed to the cAMP generating therapies formoterol, iloprost, or roflumilast, the adenylyl cyclase activator forskolin or to transforming growth factor (TGF)-b1. VEGF isoforms were evaluated with ELISA. VEGF-C release was not significantly altered by TGF-{beta}1, in contrast to the increased levels of VEGF-A, in all fibroblasts. VEGF-C was significantly decreased by iloprost, forskolin and formoterol, whereas VEGF-A was significantly increased by iloprost and forskolin, with differences in release pattern between and within fibroblasts from control and COPD subjects. Exposure to VEGF-C specifically towards VEGFR3 decreased proliferative rate in human lung fibroblasts and bronchial epithelial cells. VEGFR2 and VEGFR3 were both present in parenchymal lung tissue and VEGFR2 in pulmonary blood vessels. in both healthy and COPD, whereas there was elevated expression of VEGFR3 in bronchial epithelium. In conclusion, TGF-{beta}1 and cAMP generating compounds have significant effects on VEGF-C and VEGF-A synthesis, which appear dysregulated in lung fibroblasts from ex-smokers and patients with COPD. Increased VEGFR3 expression in the bronchial epithelium in lung tissue, and studies into their functional impact, warrants further investigations.

BackgroundThe interaction between the anti-apoptotic protein Bcl-xL and the BH3-only sensitizer BAD represents a critical regulatory checkpoint in the intrinsic apoptotic pathway. Although this interaction is known to influence mitochondrial fate, its dynamic regulation and structural determinants in living cells remain poorly understood. Here, we developed a fluorescence lifetime imaging microscopy-based Forster resonance energy transfer (FLIM-FRET) platform to visualize and quantify Bcl-xL-BAD interactions in real-time. MethodsWe developed a quantitative fluorescence lifetime-based FRET (FLIM-FRET) approach to visualize and measure Bcl-xL-BAD interactions in single living glioblastoma cells. Stable GFP/Venus-Bcl-xL and mCherry-BAD FRET pairs were created, followed by acceptor photobleaching FRET, FLIM-FRET, Annexin V-BFP-based apoptosis assays, pharmacological perturbation using BH3 mimetics, and molecular dynamics simulations with MM/GBSA analysis. Statistical significance was assessed using appropriate parametric tests across multiple independent experiments. ResultsUsing this platform, we observed that apoptotic stress markedly enhances the engagement of Bcl-xL and BAD. Increased FRET efficiency coincided with Annexin V positivity and nuclear condensation, indicating that maximal BAD binding reflects a higher level of apoptotic commitment. Structure-function analysis using targeted Bcl-xL mutants revealed distinct binding requirements: disruption of the core hydrophobic groove (Y101K) abolished BAD binding and impaired BH3 mimetic sensitivity, whereas mutation within the BH1 domain (G138A) preserved BAD interaction and sensitivity to BH3 mimetics. Molecular dynamics simulations corroborated these observations by revealing preserved BAD-binding energetics in the G138A mutant, but destabilization in the Y101K mutant. ConclusionsTogether, these findings demonstrate the utility of a live-cell FLIM-FRET platform for resolving protein-protein interactions involving apoptotic proteins at the single-cell level. By linking interaction dynamics, structural determinants, and functional outcomes, this approach provides a broadly applicable framework for studying apoptotic priming, structural tolerance at BCL-2 family interfaces, and cellular responses to BH3-mimetic therapies.

The heterotrimeric G-protein transducin (Gt) is among the key proteins mediating phototransduction in rod and cone cells of the vertebrate retina. Even though this protein has been extensively characterized in mammals, little is known about its expression patterns in migratory songbirds. Here we characterised Gt expression in the European robin, a night-migratory songbird known for its light-dependent magnetoreception. The mechanism underlying magnetoreception is not fully understood, but one well-supported hypothesis involves a radical-pair formation in the blue light receptor cryptochrome type 4a. The - and {gamma}-subunits of cone specific transducin have been identified as possible interaction partners of cryptochrome 4a. Therefore, we analysed the expression patterns of various G-protein subunits in bird photoreceptors. Specifically, we combined single cell RNA sequencing and immunohistochemistry, and tested for protein interaction by pulldown, co-immunoprecipitation, and NanoBiT luminescence assays. We show that genes for G-protein subunits GNB1 and GNB3 (coding for Gt{beta}1 and Gt{beta}3, respectively) are predominantly expressed in rods and cones. Among {gamma}-subunits, GNGT2 (coding for Gt{gamma}T2) was the principal isoform in cones, whereas GNG11 (coding for Gt{gamma}11) was associated with rods. In contrast, we did not detect GNG10 (coding for Gt{gamma}10) expression in either photoreceptor type. Interaction assays demonstrated that all three {beta}{gamma} combinations; {beta}{gamma}T2, {beta}{gamma}10, and {beta}{gamma}11, can associate in vitro. These findings indicate that {beta}{gamma} dimer formation in vivo is likely constrained by the photoreceptor-specific expression of the respective subunits. Furthermore, the absence of GNG10 expression in rods and cones does not support a role of this {gamma}-subunit in photoreceptor-based magnetoreception.

Ionizing radiation induces DNA damage and oxidative stress; however, the genes and molecular mechanisms involved in bacterial stress responses have not been sufficiently identified. In this study, we used Limnobacter thiooxidans strain CS-K2, which is the closest relative to the bacteria detected in torus room water at the Fukushima Daiichi Nuclear Power Plant according to 16S rRNA gene sequences, and evaluated its response to {gamma}-ray irradiation using integrated transcriptomic and proteomic analyses. We identified three previously uncharacterized genes (LT3105, LT3115, and LT3126) that were strongly induced at the mRNA and protein levels. These genes exhibited low basal expression but were markedly upregulated by {gamma}-ray irradiation. Notably, LT3126 encodes a protein containing VIT (vault protein inter--trypsin) and VWA (von Willebrand factor type A) domains and showed the strongest induction. Overexpression of LT3126 increased survival after 500 Gy irradiation by approximately 200-fold compared with the control bacteria, demonstrating a direct contribution to survival under high-dose stress. Comparative genomic analysis showed that these genes are not widely conserved across bacteria but are unevenly distributed among specific lineages. Taken together, this study identified a novel set of {gamma}-ray-responsive genes and demonstrated a functional role for LT3126 in radiation resistance, providing new insights into molecular adaptation in radiation-associated environments. IMPORTANCEWe identified a novel set of {gamma}-ray-responsive genes (LT3105, LT3115, and LT3126) in the non-model bacterium Limnobacter thiooxidans. These genes are located in relatively close genomic proximity and are coordinately induced upon irradiation, suggesting a shared functional role in stress response. Overexpression of LT3126 increased survival by approximately 200-fold after 500 Gy irradiation compared with the control bacteria, demonstrating a substantial contribution to survival under high-dose stress. These genes were also induced by heat shock and oxidative stress, indicating that their function extends beyond radiation-specific responses to broader environmental stress adaptation. Consistent with this, comparative genomic analysis showed that these genes are not widely conserved across bacteria but are unevenly distributed among specific lineages. Taken together, these findings highlight previously unrecognized molecular strategies that may support bacterial survival in radiation-associated environments.

Microproteins are proteins of <100 amino acids. They represent a major, and until recently, overlooked fraction of the human proteome. However, it has now been demonstrated that many of these proteins play key roles in cellular physiology. Our group reported the expression of a microprotein expressed from an ioORF within the 53BP1 CDS arising as a result of delayed translational reinitiation mediated by a small uORF within the 5 TL. We named this microprotein SEP53BP1. We have sought to expand these studies with the ultimate aim of establishing a function for this microprotein. Although this remains elusive, we report findings providing new insights into the elements regulating its translation and demonstrate that the SEP53BP1 sequence serves as a Golgi targeting tag. Lastly, despite the fact that subunits of the proteasome feature prominently on interactome studies we were unable to demonstrate an impact of microprotein over-expression on the activities of both the proteasome and immunoproteasome.