International Journal of Biological Macromolecules

Proteases are an important class of proteolytic enzymes having great importance in both basic science and industrial applications. While cells tightly regulate the spatio-temporal activity of different proteases for cellular homeostasis, mis-regulation often leads to adverse effects. In this context, the protease activity of papain and its activation by L-cysteine is poorly understood in the literature. Herein, we discover that the protease activity of papain can be effectively regulated via a spontaneous liquid-liquid phase separation (LLPS) pathway. We show that papain undergoes biomolecular condensation via spontaneous LLPS under macromolecular crowding through the involvement of intermolecular hydrophobic interactions. Secondary structure analyses revealed a compact conformation of phase-separated papain with increased -helix content. Although native free papain is found to be active towards synthetic and protein substrates, the proteolytic digestion produces heterogeneous peptide aggregates. In contrast, we found that papain droplets remain dormant toward protein digestion due to the disulfide linkage of the active cysteine residue (Cys-25) in its compact conformational state. More importantly, we show that the protease activity of phase-separated papain can be reactivated in the presence of L-cysteine to produce uniform soluble peptide fragments. Our findings indicate that although disulfide linkages are not necessary for the phase separation of papain, upon phase separation, intermolecular interactions between phase-separated papain result in the formation of disulfide linkages involving active Cys-25 residues. The present discovery has tremendous technological importance to boost the efficacy of meat tenderization in the food industry.

Antifreeze proteins (AFPs) found in various cold-adapted organisms inhibit ice growth and are of interest for applications in food products, cryopreservation, agriculture, and materials science. Although high-resolution structures are available for several AFPs, the amino acids required for full antifreeze activity remain incompletely defined, and the development of AFP variants with properties such as enhanced solubility, high expression yield, and improved thermostability may further facilitate applications. Here, we used the deep learning model ProteinMPNN to redesign the globular fish antifreeze protein AFPIII, keeping the previously reported ice-binding residues fixed. We readily obtained sequences confidently predicted to adopt AFPIIIs structure and we selected five designed variants for expression, all of which expressed efficiently in E. coli. Circular dichroism spectroscopy showed that two of these variants retained secondary structure elements consistent with AFPIII, whereas the other three exhibited structural differences. One design was predicted and experimentally confirmed to have increased thermostability. All five variants displayed measurable thermal hysteresis activity. However, none reached the activity of wild-type AFPIII, suggesting that maintaining the currently established set of ice-binding residues is not sufficient to fully preserve this AFPs function; other, unidentified residues can also impact its activity. Our findings highlight the value of deep learning-based protein design methods both for generating AFP variants with desirable properties and for uncovering gaps in existing knowledge of well-characterized AFPs.

Amyloid fibrils are traditionally associated with protein misfolding disorders; however, increasing evidence indicates that they can also perform beneficial biological functions, including antimicrobial defense. Here, we investigated whether structurally distinct amyloid polymorphs of hen egg white lysozyme (HEWL) exhibit enhanced antibacterial activity compared with the native protein. HEWL was converted into two amyloid polymorphs, flexible fibrils (FFs) and rigid fibrils (RFs), and their antibacterial activities were evaluated against the Gram-positive bacterium Staphylococcus aureus and the Gram-negative bacteria Escherichia coli (Top10) and Salmonella Typhimurium. Fibril formation was confirmed by circular dichroism (CD) spectroscopy, thioflavin T (ThT) fluorescence, and transmission electron microscopy (TEM), demonstrating morphologically distinct amyloid assemblies with different secondary-structure organizations. Fluorescence-based bacterial growth assays showed that native HEWL exhibited only moderate antibacterial activity, whereas both amyloid polymorphs produced potent, concentration-dependent bacterial growth inhibition. FFs and RFs consistently displayed greater antibacterial efficacy than native HEWL across all tested strains, with FFs exhibiting slightly stronger activity against S. Typhimurium. At concentrations of 600-800 M, FFs achieved >90% growth inhibition for all bacterial species examined. Cytotoxicity studies using SH-SY5Y human neuroblastoma cells demonstrated minimal toxicity for native HEWL, modest effects for FFs, and substantially greater toxicity for RFs, indicating that amyloid polymorphism influences both antimicrobial activity and mammalian cell compatibility. Collectively, these findings establish a direct relationship between amyloid structure, antibacterial efficacy, and cytotoxicity. The combination of potent antibacterial activity and relatively low cytotoxicity identifies FFs as a promising functional amyloid biomaterial for the development of next-generation antimicrobial materials.

The widespread use of synthetic food dyes, such as Acid Yellow 23 (AY 23), in the food, cosmetics, and pharmaceutical industries raises questions about their potential effects on biological systems and public health. The concentration-dependent interaction between AY 23 and bovine serum albumin (BSA), a crucial model protein for understanding pharmacokinetics and protein-ligand behaviour, was examined in this study. We demonstrate that, under physiological conditions, increasing dye concentrations from 50 M to 200 M results in notable conformational changes, increased surface hydrophobicity, and protein aggregation using a multimodal biophysical approach that includes fluorescence spectroscopy. Direct visualisation verified these structural changes and aggregate formation, whereas hemolytic assay confirmed the high hemolytic nature of AY 23-induced fibrils. Additionally, this study provides a mechanistic basis for the toxicological effects of AY 23, underscoring the implications of food dyes for public health.

LIM kinases, LIMK1 and LIMK2, play a crucial role in cytoskeleton dynamics. They are involved in many physiological processes but also in several pathologies such as cancer, neuronal diseases and neurofibromatosis. Although LIM kinases appear as promising therapeutic targets, they remain undruggable. A better understanding of their activity and regulation is thus required to better design efficient targeted therapies. Here, we have shown the impact of a single amino acid on LIMK activity on cofilin, their main substrate in actin filament remodelling. We demonstrated that Y632 and Y630, for LIMK1 and LIMK2 respectively, mediate LIMK dimerization, resulting in their transphosphorylation. This process seems to be a prerequisite for their canonical phosphorylation on their respective T508 and T505 residues within the activation loop. These Tyrosine are not phosphorylated, their aromatic nature is rather critical to ensure proper LIMK activity on cofilin. These results bring new insights into LIMK molecular features.

Glycation, a non-enzymatic reaction occurring between sugars and biological macromolecules, plays a critical role in ageing and disease pathogenesis. Methylglyoxal (MG) is a highly reactive -oxoaldehyde that leads to the formation of endogenous advanced glycation end products (AGEs). These AGEs are associated with diabetes and many other diseases, including neurodegeneration and cancer. This is often through interactions with the receptor for advanced glycation end products (RAGE). Inhibition of glycation/AGEs formation using natural products to target cancer is an area of recent interest. In vitro AGEs formation was observed by browning of samples, increased fluorescence, and carbonyl stress. MG induced changes in the structure of BSA were analysed using electrophoresis, spectroscopy, TEM, AFM, DLS, and CD spectroscopy. Our results show that AGEs form random structures, oligomeric aggregates, and {beta}-sheets. Thioflavin T and Congo red staining further validated these findings. Galangin and Caffeic acid demonstrated significant antiglycation activity, suppressing AGEs formation in vitro. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=134 SRC="FIGDIR/small/737425v1_ufig1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@113b391org.highwire.dtl.DTLVardef@7208a1org.highwire.dtl.DTLVardef@94c2e1org.highwire.dtl.DTLVardef@867b85_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIMethylglyoxal-induced Advanced Glycation End Products were prepared in vitro C_LIO_LIMethylglyoxal -induced structural modifications in BSA C_LIO_LIAGEs were characterised using various parameters C_LIO_LIBoth fluorescent and non-fluorescent AGEs were formed. C_LIO_LIPhytochemical treatment induced inhibition of AGEs formation C_LI

Silk proteins, including sericin and fibroin, are natural biopolymers with broad applications in tissue engineering where angiogenesis plays an essential role. However, the pro-angiogenic effects of silk proteins with varying molecular weights (MWs) remain poorly understood. Here, silk proteins with MW distributions at 40-180 kDa or less than 25 kDa were obtained through alkaline hydrolysis to evaluate their effects on angiogenesis. Structurally, reducing MW induced a conformational transition in silk proteins, accompanied by a striking morphological shift in sericin from nanofibers to nanoparticles. Functionally, high-MW sericin (SSH) suppressed, whereas low-MW sericin (SSL) and both high- and low-MW silk fibroin (SFH/SFL) directly promoted endothelial angiogenic activity. Transcriptomic analysis revealed that angiogenesis-related genes such as Id1 and Smad6/9 may underlie the angiostatic effects of SSH. Notably, both SSH and SSL enhanced angiogenesis indirectly via macrophages; however, SSH induced mixed M1/M2-like polarization, while SSL preferentially drove an M2-like phenotype. In a subcutaneous implantation model, SSH promoted angiogenesis but yielded vessels with weak integrity and increased fibrosis, whereas SSL enhanced angiogenesis with improved vascular maturity and reduced fibrotic response. These findings elucidate how the MWs of silk proteins shape angiogenic behavior and highlight the importance of MW tailoring for optimized tissue engineering applications.

Prion diseases are neurodegenerative disorders associated with the structural conversion of the cellular prion protein (PrPc) into its misfolded infectious isoform (PrPSc). Despite substantial efforts, no disease-modifying therapy or cure is currently available. Here, we present an integrated computational-experimental pipeline for the rational design of cyclic peptides targeting PrPc to inhibit its pathogenic conversion. Starting from crystal structures of antibody-bound mouse PrPc, we develop a rational design strategy combined with iterative molecular dynamics simulations and sequence optimization to generate peptides with enhanced binding and structural impact. Three candidates were selected for experimental validation. Our results show that PH1 (49YGPDPSDSYT58, antibody numbering) that binds stably to the &alpha2-&alpha3 interface most effectively reduced PrPSc levels in GT1-7 cells, essentially by inducing allosteric rearrangements that reinforce the intramolecular helical bundle. PL1 (89GQSNTKPYT97) and PL2 (89RQSNTWPYT97) binding the &beta1-&alpha1/&alpha3 junction exerted more modest effects due to the potential competition of the flexible tail to bind at this site. These results establish a mechanistic link between peptide-induced stabilization of PrPc and inhibition of prion propagation and provide a generalizable framework for designing conformational stabilizers of aggregation-prone proteins.

Introduction: Thromboinflammation, which represents the pathological interplay between inflammation and thrombosis, is a leading cause of global mortality. Current therapies are frequently associated with an increased risk of bleeding and do not adequately address the inflammatory component of the disease. The African tree Guibourtia tessmannii represents a promising source of natural anti-inflammatory compounds. This study aimed to synthesize and characterize silver nanoparticles using an aqueous bark extract of G. tessmannii (GT-AgNPs) and to evaluate their anti-inflammatory and anticoagulant properties. Methods: GT-AgNPs were synthesized by reducing silver nitrate with an aqueous extract of G. tessmannii bark. The nanoparticles were comprehensively characterized using UV-Vis spectroscopy, FTIR spectroscopy, powder X-ray diffraction, and scanning electron microscopy. In vitro anti-inflammatory activity was evaluated through inhibition of bovine serum albumin denaturation, whereas in vivo anti-inflammatory activity was assessed using the carrageenan-induced rat paw edema model. Anticoagulant activity was investigated by measuring activated partial thromboplastin time (aPTT) and prothrombin time (PT), corresponding to the intrinsic and extrinsic coagulation pathways, respectively. Results: The synthesis successfully produced GT-AgNPs with an average particle size of approximately 20 nm. Both the aqueous extract and GT-AgNPs exhibited marked anti-inflammatory activity. The nanoparticles achieved 95% inhibition of protein denaturation in vitro and 95% inhibition of carrageenan-induced paw edema in vivo at a dose of 0.4 mg/kg body weight after 5 h. Furthermore, both the extract and GT-AgNPs demonstrated dose-dependent anticoagulant activity. Conclusion: The study demonstrated that GT-AgNPs, synthesized from the bark of G. tessmannii, possess significant anti-inflammatory and anticoagulant properties. These findings highlight the potential of GT-AgNPs as nanotherapeutic candidates for the management of thrombo-inflammatory disorders.

Drug-resistant tuberculosis and the rising incidence of nontuberculous mycobacterial (NTM) infections are a growing concern that demands innovative therapeutic strategies. Despite advances in diagnostics, drug discovery, and vaccine strategies, significant gaps remain. Mycobacteriophages and their lytic enzymes offer a promising solution due to their natural abundance and diversity, host specificity and ability to disrupt complex cell envelopes and biofilms. In this study, we report the genomic and functional characterization of a V-Cluster mycobacteriophage, EniyanLRS, isolated near a hospital in Delhi and the encoded endolysins LysA and LysB. EniyanLRS features a 78.53 kbp genome with a notably low GC content (56.9%) as compared to other mycobacteriophages, and an exceptionally long Tape Measuring Protein (TMP) gene (5.97 kbp). Its genome lacks genes related to lysogeny and harbours 24 tRNAs, suggesting high translational efficiency. Phenotypically, EniyanLRS exhibits a siphovirus morphology, lytic lifecycle and infects Mycobacterium smegmatis and drug-resistant Mycobacterium fortuitum. LysA, with its lysozyme-chitinase-amidase domain architecture, did not demonstrate significant antibacterial or antibiofilm activity. Conversely, LysB, an /{beta}-hydrolase, exhibited superior in vitro esterase activity compared to previously reported LysB enzymes and showed pronounced cell wall disruption of M. smegmatis and M. fortuitum, along with considerable antibiofilm efficacy (62.77% and 41.91% inhibition, respectively). Collectively, these findings highlight the potential of EniyanLRS and its LysB enzyme as potent biocontrol agents against pathogenic mycobacteria, which can be explored to treat planktonic cells and biofilm-associated infections.

The growing number of approved nucleic acid therapeutics illustrates the potential to treat diseases by targeting their genetic blueprints in vivo. The 10-23 DNAzyme is capable of cleaving a wide range of target RNA with high selectivity. However, its poor performance in vivo restricts its therapeutic application as gene silencing agent. Studies on ribozymes have shown that the crowded environment in cells and associated effects can impact ribozyme folding and thermostability, resulting in a change in activity. This opens up the question whether DNAzymes are also affected by molecular crowding. Here, we investigate the functional and structural influence of molecular crowding conditions on the 10-23 DNAzyme. The stability and activity of a PrP-specific 10-23 DNAzyme were examined in presence of PEG, dextran, and osmolytes. Our results indicate that osmolytes decrease DNAzyme activity in a concentration-dependent manner, while certain PEG and dextran concentrations promote activity. To rationalize our observations, we studied the cosolutes effect on physicochemical solution properties and the structure of the DNAzyme:RNA complex using FCS and SAXS. The data reveal that enhanced activity is observed under conditions where a combination of physiochemical properties matches an optimum that seems to be dependent on the metal ion cofactor. Structural influence under such conditions is indicated less. We propose that a certain degree of molecular crowding is required to favor a state, which allows for higher catalytic turnover. In addition, we show that the requirement for magnesium and manganese as a cofactor remains unchanged under the conditions applied. Our work contributes to a better understanding of how the cellular environment affects DNAzyme structure and function.

CHI3L1 (YKL-40) is a chitinase-like glycoprotein involved in immune regulation, tissue remodeling, and cancer, yet the molecular principles governing its glycan interactions remain incompletely defined. Previous reports suggested that CHI3L1 can bind to chitin oligosaccharides (COS) and glycosaminoglycan (GAG) ligands, however, the molecular basis and binding sites underlying these interactions remain controversial. Here, a combination of biophysical and computational methods is employed to shed light on carbohydrate interactions of the protein and delineate a potential crosstalk between its glycan-binding interfaces. Our results demonstrate that COS and GAGs bind to distinct, non-overlapping sites on CHI3L1. Both ligand classes exhibit a strong dependence of binding affinity on the degree of polymerization. Molecular dynamics simulations, supported by mutational analysis, identify a GAG-binding site centered on residues R144, R145, and K147 and reveal an additional distal interaction site for longer GAG ligands. Biophysical and biochemical assays fail to confirm a previously proposed allo- or orthosteric interaction between both binding sites. However, physiologically relevant protein-protein interactions mediated by the chitin binding site of CHI3L1 are differentially regulated by GAG and COS ligands. COS inhibit binding of galectin-3 to CHI3L1, whereas GAG ligands enhance the affinity between the proteins by ca. 14-fold. Together, these findings establish CHI3L1 as a dual carbohydrate-binding protein with distinct recognition interfaces and reveal a previously unrecognized role for GAGs in modulating CHI3L1-mediated signaling interactions.

Glyceraldehyde (GA) contributes to the development of various diseases, such as diabetes and Alzheimer's disease via protein glycation and the formation of advanced glycation end products (AGEs); however, effective strategies for neutralizing GA are limited. Carnosine (Car), an imidazole dipeptide (IDP) that is abundant in meat, suppresses protein glycation by scavenging reactive aldehydes. There are only a few reports on the antiglycation activity of Car against GA. For other IDPs, such as anserine, balenine (Bal), and homocarnosine, there are almost no reports on their antiglycation activity. In this study, we demonstrated the antiglycation activity of four types of IDPs and 2-oxocarnosine (2-oxo-Car), an oxidized form of Car, against GA-induced intracellular protein glycation and neuronal cytotoxicity. Car and Bal exhibited significantly higher reactivity with GA compared with other IDPs and 2-oxo-Car. An in silico analysis suggested that the difference in reactivity is dependent upon intramolecular hydrogen bond formation and the conformation of each IDP. Although there were differences in reactivity with GA, LC-MS analysis revealed that all of the IDPs and 2-oxo-Car reacted with two molecules of GA to form adducts containing pyridinium rings. Car and Bal exhibited high reactivity with GA and markedly suppressed GA-induced cytotoxicity in SH-SY5Y cells. Western blot and qPCR analyses revealed that IDPs suppressed GA-induced protein glycation and the upregulation of endoplasmic reticulum and oxidative stress response genes. Our results indicate that IDPs represent a novel preventive approach to AGE-related diseases and provide a foundation for the development of strategies to treat GA-related neurotoxicity.

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.

Dengue Virus Serotype 2 is a human pathogenic flavivirus that encodes a non-structural protein 3 (DEN2-NS3) containing a helicase domain essential for viral replication. DEN2-NS3 utilizes energy derived from NTP hydrolysis to unwind dsRNA and dsDNA. A galloylated catechin, (-)-epigallocatechin gallate (EGCG), was previously reported to be highly potent against the Zika Virus NS3 helicase, with an IC50 value observed at 295.7 nM. This prompted an investigation to determine if three catechins, namely, (-)-epigallocatechin (EGC), (-)-epicatechin gallate (ECG), and EGCG, would act as potent inhibitors of DEN2-NS3. Enzyme-inhibition assays revealed that the helicase catalytic domain, DEN2-NS3(S171-K618), is strongly inhibited by these galloylated catechins. We observed Ki values of 400 {+/-} 86.6 nM for EGCG (mixed-mode inhibition with respect to ATP) and 550 {+/-} 250 nM for ECG (uncompetitive inhibition with respect to ATP). Furthermore, using a computational workflow starting with SiteMap, we provide evidence that a highly druggable pocket exists within the RNA-binding cavity, involving residues ASP290, ARG387, ASP409, MET429, HIS487, ASP541, ARG599, and ASP603. These catechins were each analyzed through 200-ns molecular dynamics (MD) simulations to evaluate the binding stability within the target DEN2-NS3 binding pocket. Computational results revealed that EGCG and ECG maintained high stability, forming shared, highly persistent amino acid contacts (>45% occupancy) with ASP603, ARG599, ASP541, and ARG387. In conclusion, we have demonstrated that EGCG and ECG achieve strong binding and allosteric disruption of the critical RNA-binding channel. We suggest that future structural optimization of these compounds into stable prodrug derivatives could yield promising antiviral therapies. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=99 SRC="FIGDIR/small/733882v1_ufig1.gif" ALT="Figure 1"> View larger version (41K): org.highwire.dtl.DTLVardef@2db363org.highwire.dtl.DTLVardef@5c2fdaorg.highwire.dtl.DTLVardef@49bf8eorg.highwire.dtl.DTLVardef@1bf31f1_HPS_FORMAT_FIGEXP M_FIG C_FIG

Herpes simplex virus type 1 (HSV-1) is a common human pathogen that undergoes lytic replication in epithelial and other permissive cell types and can establish latency in peripheral neurons. ICP22 is a multifunctional HSV-1 immediate-early protein that localizes to the nucleus of infected cells; however, its interactions with host cellular factors remain incompletely understood. Here, ICP22 was demonstrated to interact with the human antisense function 1 protein (ASF1), including both ASF1a and ASF1b, in transfected cells and HSV-1-infected cells, respectively. ICP22 also colocalized with ASF1 in the nucleus. ICP22 amino acids 213 to 340 are important for the interaction of ICP22 with ASF1, whereas amino acids 37 to 153 of ASF1a and ASF1b are critical for their interactions with ICP22. Furthermore, ICP22 expression was associated with reduced ASF1-H3.1 co-immunoprecipitation under the tested conditions. ASF1 knockdown also reduced HSV-1-BAC-Luc luciferase output, indicating that ASF1 contributes to efficient infection-associated reporter activity in this study. Collectively, these results indicate that the interaction of HSV-1 ICP22 with ASF1 might help regulate the transcription of viral or cellular genes during HSV-1 infection. Keywords: HSV-1, ICP22, ASF1, histone H3.

The phenotypic plasticity, slow replication, and complex, hydrophobic cell envelope of Mycobacterium tuberculosis contribute to its successful survival as a pathogen and its drug tolerance. Consequently, the global threat of multidrug-resistant Tuberculosis (MDR-TB), coupled with lengthy and highly toxic treatment regimens, necessitates the development of innovative treatment solutions. Mycobacteriophages are natural viruses of mycobacteria that typically encode two endolysins, which cooperatively facilitate host cell lysis at the end of the lytic life cycle: LysA, a peptidoglycan hydrolase, and LysB, a lipolytic enzyme, targeting the mycolylarabinogalactan-peptidoglycan complex. Their precise and efficient lytic activity, along with their low propensity to induce resistance, make them, particularly LysBs, promising candidates for new treatment solutions. In this study, we report MTB-LysB1, a novel LysB enzyme from an F1 sub-cluster mycobacteriophage isolated from our laboratory collection. While studying its structural features by comparing the modelled structure with representative mycobacteriophage LysB homologues, we found that the /{beta}-hydrolase fold and key motifs are conserved. Also, we identified putative membrane-interaction motifs that may play a role in LysB1s cell permeation. Significantly, we found MTB-LysB1 to be active against both drug-susceptible and multidrug-resistant (MDR) M. tuberculosis strains at nanomolar concentrations, comparable to the well-characterised D29 LysB reference enzyme. Beyond its standalone activity, MTB-LysB1 exhibits an additive effect when combined with the TB drugs rifampicin and moxifloxacin, and co-administration reduces the drugs minimum inhibitory concentrations (MICs), which holds clinical significance. By structurally damaging the mycobacterial cell wall, the enzyme appears to act as a permeability enhancer for the chemotherapeutic drugs, thereby improving antibiotic efficacy. Collectively, our findings position the enzyme not only as a novel antimycobacterial agent but also provide a structural framework for its rational engineering as a promising next-generation adjunct to TB drug regimens. HighlightsO_LIA novel F1 sub-cluster phage-derived LysB is discovered and characterised using integrated computational, biochemical and microbiological methods. C_LIO_LIAlphaFold2 modelling, molecular dynamics simulations and comparative structural analyses revealed an /{beta}-hydrolase fold with conserved catalytic and membrane-interaction features. C_LIO_LIThe enzyme exhibited high esterase activity, thermal stability and potent lytic activity against Mycobacterium tuberculosis. C_LIO_LIAn additive effect with TB drugs rifampicin and moxifloxacin highlights MTB-LysB1s potential as an adjunct therapeutic. C_LI

Cnidarians possess large number of opsins in their genomes for their various photoreceptive functions. In particular, they uniquely possess Gs-coupled opsins that induce intracellular cAMP accumulation in a light-dependent manner. These Gs-coupled opsins, cnidopsins, are powerful optogenetic tools manipulating cAMP-dependent cellular responses. In this study, we characterized a cnidopsin, named as AtCnidop3a, from the coral Acropora tenuis as a Gs-coupled and UV-sensitive bistable pigment. This cnidopsin showed a large spectral shift upon activation from absorption maxima from 395 nm to 560 nm, and the resting and activated states are interconvertible by illumination with UV (or violet) and orange light. The activated state efficiently activated Gs proteins and elevated intracellular cAMP levels in mammalian cultured cells. To engineer the opsin mutant that can be turned on and off upon long wavelength light illumination by utilizing the large spectral separation, negatively charged amino acids were introduced near the retinal Schiff base region. Among tested opsin mutants, the Y1133.28E mutant is capable of being activated by green light unlike the wild-type while retaining the property of being inactivated by orange light like the wild-type, indicating successful conversion of the opsin to a visible light sensitive bistable pigment. The visible light-induced cAMP regulation of the Y1133.28E mutant was enhanced by an additional L942.61G substitution. Our characterization and engineering of the cnidopsin revealed functional diversity of cnidarian opsins and its potential utility as optogenetic tools regulating Gs-dependent physiological responses.

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.

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.