Search Results (309)

TasA gene, encoding a functional amyloid protein critical for biofilm formation and antimicrobial activity, was cloned from the endophytic strain Bacillus amyloliquefaciens BS-3, isolated from rubber tree roots. This study identified the shortest functional TasA variant (483 bp, 160 aa) reported to date, featuring unique amino acid substitutions in conserved domains. Bioinformatics analysis predicted a signal peptide (1–27 aa) and transmembrane domain (7–29 aa), which were truncated to optimize heterologous expression. Two prokaryotic vectors (pET28a and pCZN1) were constructed, with pCZN1-TasA expressed solubly in Escherichia coli Arctic Express at 15 °C, while pET28a-TasA formed inclusion bodies at 37 °C. Purified recombinant TasA exhibited potent antifungal activity, achieving 98.6% ± 1.09 inhibition against Colletotrichum acutatum, 64.77% ± 1.34 against Alternaria heveae. Notably, TasA completely suppressed spore germination in C. acutatum and Oidium heveae Steinmannat 60 μg/mL. Structural analysis via AlphaFold3 revealed that truncation enhanced protein stability. These findings highlight BS-3-derived TasA as a promising biocontrol agent, providing molecular insights for developing protein-based biopesticides against rubber tree pathogens. Full article

Low-temperature stress severely limits plant growth and reduces agricultural productivity. Calmodulin-like (CML) proteins are crucial calcium sensors in plant cold responses. Transcriptome analysis of cold-stressed Saussurea involucrata identified seven differentially expressed CML genes. qRT-PCR confirmed that SiCML6 was strongly induced at 4 °C and −2 °C. Bioinformatics analysis showed that SiCML6 encodes a transmembrane protein containing an EF-hand domain. This protein carries a signal peptide and shows the closest phylogenetic relationship to Helianthus annuus CML3. Its promoter contains ABA, methyl jasmonate (MeJA), and cold-response elements. Arabidopsis plants overexpressing SiCML6 showed significantly higher survival rates at −2 °C than wild-type plants. Under freezing stress, SiCML6-overexpressing lines exhibited reduced malondialdehyde content, relative electrolyte leakage, and ROS accumulation (H₂O₂ and O₂⁻), along with increased proline, soluble sugars, soluble proteins, and total antioxidant capacity (T-AOC). SiCML6 elevated the expression of cold-responsive genes CBF3 and COR15a under normal conditions and further upregulated CBF1/2/3 and COR15a at 4 °C. Thus, low temperatures induced SiCML6 expression, which was potentially regulated by ABA/MeJA. SiCML6 enhances freezing tolerance by mitigating oxidative damage through boosted T-AOC and osmoprotectant accumulation while activating the CBF-COR signaling pathway. This gene is a novel target for improving crop cold resistance. Full article

Background: The introduction of cystic fibrosis transmembrane conductance regulator modulators, especially the triple therapy elexacaftor, tezacaftor, ivacaftor (ETI), has improved outcomes in people with cystic fibrosis (pwCF), reducing underweight but increasing overweight rates. Objectives: This study investigates the effect of ETI on appetite control, body composition, and energy balance during a 3-week inpatient rehabilitation programme with regular exercise. Methods: In 54 pwCF (38 on ETI, 16 without ETI), changes in body composition (fat mass index, FMI; fat-free mass index, FFMI) and energy balance (calculated from body composition changes) were assessed. Appetite control was evaluated via plasma peptide YY (PYY) levels and post-exercise meal energy intake. Results: The programme significantly increased BMI (+0.3 ± 0.1 kg/m²; CI 0.1–0.4) and energy balance (+4317 ± 1976 kcal/3 weeks), primarily through FFMI gains (+0.3 ± 0.1 kg/m²; CI 0.1–0.4). Despite higher post-exercise meal energy intake and a tendency towards lower PYY levels in the ETI group, changes in body composition and energy balance did not differ between groups. This is explained by a higher prevalence of exocrine pancreatic insufficiency in the ETI group (92% vs. 50%, p < 0.001). Small sample sizes limit the interpretation of data on appetite control and energy intake. Conclusions: A 3-week inpatient rehabilitation programme improved body composition in pwCF, without resulting in a more positive energy balance with ETI therapy. This is due to a higher prevalence of pancreatic insufficiency in this group. Full article

Cold environments challenge the structural and functional integrity of membrane proteins, requiring specialized adaptations to maintain activity under low thermal energy. Here, we investigate the molecular basis of cold tolerance in the peptide transporter PepT1 from the Antarctic icefish (Chionodraco hamatus, ChPepT1) using molecular dynamics simulations, binding free energy calculations (MM/GBSA), and dynamic network analysis. We compare ChPepT1 to its human ortholog (hPepT1), a non-cold-adapted variant, to reveal key features enabling psychrophilic function. Our simulations show that ChPepT1 displays enhanced global flexibility, particularly in domains adjacent to the substrate-binding site and the C-terminal domain (CTD). While hPepT1 loses substrate binding affinity as temperature increases, ChPepT1 maintains stable peptide interactions across a broad thermal range. This thermodynamic buffering results from temperature-sensitive rearrangement of hydrogen bond networks and more dynamic lipid interactions. Importantly, we identify a temperature-responsive segment (TRS, residues 660–670) within the proximal CTD that undergoes an α-helix to coil transition, modulating long-range coupling with transmembrane helices. Dynamic cross-correlation analyses further suggest that ChPepT1, unlike hPepT1, reorganizes its interdomain communication in response to temperature shifts. Our findings suggest that cold tolerance in ChPepT1 arises from a combination of structural flexibility, resilient substrate binding, and temperature-sensitive interdomain dynamics. These results provide new mechanistic insight into thermal adaptation in membrane transporters and offer a framework for engineering proteins with enhanced functionality in extreme environments. Full article

The recent global coronavirus pandemic highlighted the ever-present threat of respiratory virus outbreaks and the consequent need for ongoing research into antiviral therapy. To this end, structural analogues of the guanidinium-based drug camostat mesylate have been synthesised to probe their potential inhibition of Transmembrane Serine Protease 2 (TMPRSS2), a human protease that is essential for infection by many respiratory viruses, including Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). Our in vitro fluorescence-based protease assays and supporting computational docking studies suggest that C-terminal camostat analogues retain TMPRSS2 inhibition potencies (IC₅₀ = 1–3 nM, BE = −6.6 to −7.0 kcal/mol) that match or exceed that of the parent drug. Analogues 1c and 1d emerge as lead candidates in this regard, thereby validating the rationale behind C-terminal structural modifications and highlighting these derivatives as promising scaffolds for the future development of targeted antiviral therapeutics. Replacement of camostat’s ester functionality with peptide linkages largely preserves non-covalent binding but disrupts in vitro protease inhibition, findings consistent with the parent drug’s known role as an acylating suicide inhibitor. Docking studies confirm that the replacement of aromatic residues with flexible, equivalent-length alkyl chains is detrimental to drug binding. These function and binding data offer new directions for the synthesis of further analogues of camostat and of other guanidinium-based protease inhibitors that have yet to be refined via structure–activity relationship studies. Further investigation will support tailoring this class of drugs for repurposing in antiviral therapy. Full article

Angiotensin-converting enzyme 2 (ACE2) is a cell-surface receptor that helps the body regulate blood pressure and endocrine secretions. Transmembrane serine protease 2 (TMPRSS2) is a cell surface protein expressed mainly by endothelial cells of the respiratory and digestive tract, which participates in the cleavage of protein peptide bonds with serine as the active site. These two proteins have been studied to be highly associated with infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Soybean trypsin inhibitor (SBTI) has special bioactivities such as anticarcinogenic and anti-inflammatory functions, which can be widely used in functional foods or drugs. Our study involved in vitro and in vivo experiments to elucidate the effect of SBTI on SARS-CoV-2 host invasion. First, it was confirmed that being under 250 μg/mL of SBTI was not toxic to HepG2, HEK293T, and Calu-3 cells. The animal study administered SBTI to mice once daily for 14 days. In the lungs, liver, and kidneys, the histopathologic findings of the SBTI group were not different from those of the control group, but the expression of ACE2, TMPRSS2, and CD147 was reduced. Thus, our findings suggest that the inhibition of ACE2, TMPRSS,2 and CD147 proteins by SBTI shows promise in potentially inhibiting SARS-CoV-2 infection. Full article

Tomato chlorosis virus (ToCV) is a highly infectious plant virus that poses a significant threat to the Solanaceae family worldwide. Despite its widespread impact, effective control remains challenging due to its vector-borne transmission by whiteflies. To facilitate early detection and potential therapeutic intervention, this study aimed to identify diagnostic epitopes through a comprehensive bioinformatics approach combining comparative genomics and artificial intelligence-based structural modeling. We analyzed forty-four complete ToCV genomes to identify highly conserved regions and uncovered an orphan clade, indicating evolutionary divergence. Subcellular localization and transmembrane domain predictions revealed viral proteins with extracellularly exposed peptide regions. Structural modeling using AlphaFold3 further validated the stability and accessibility of these domains. By integrating these findings with epitope prediction algorithms, this study identified four highly promising epitope candidates, which are suitable for the development of antibody-based diagnostic kits and antiviral therapeutics targeting ToCV. These epitopes provide a strong foundation for the development of antibody-based diagnostic kits or antiviral therapeutics targeting ToCV. Full article

TRPA1 (Transient Receptor Potential Ankyrin 1), a cation channel predominantly expressed in sensory neurons, plays a critical role in detecting noxious stimuli and mediating pain signal transmission. As a key player in nociceptive signaling pathways, TRPA1 has emerged as a promising therapeutic target for the development of novel analgesics. Crotalphine (CRP), a 14-amino acid peptide, has been demonstrated to specifically activate TRPA1 and elicit potent analgesic effects. Previous cryo-EM (cryo-electron microscopy) studies have elucidated the structural mechanisms of TRPA1 activation by small-molecule agonists, such as iodoacetamide (IA), through covalent modification of N-terminal cysteine residues. However, the molecular interactions between TRPA1 and peptide ligands, including crotalphine, remain unclear. Here, we present the cryo-EM structure of ligand-free human TRPA1 consistent with the literature, as well as TRPA1 complexed with crotalphine, with resolutions of 3.1 Å and 3.8 Å, respectively. Through a combination of single-particle cryo-EM studies, patch-clamp electrophysiology, and microscale thermophoresis (MST), we have identified the cysteine residue at position 621 (Cys621) within the TRPA1 ion channel as the primary binding site for crotalphine. Upon binding to the reactive pocket containing C621, crotalphine induces rotational and translational movements of the transmembrane domain. This allosteric modulation coordinately dilates both the upper and lower gates, facilitating ion permeation. Full article

Signal peptides (SPs) are short amino acid sequences located at the N-terminus of nascent proteins and are widely present across various life forms. They play crucial roles in protein synthesis, transmembrane transport, and intracellular signal transduction. With the rapid advancement of bioinformatics, studies have revealed that the functions of SPs are far more complex than previously understood. In recombinant protein expression systems, the rational design and optimization of SPs are essential for enhancing the expression efficiency and secretion level of exogenous proteins. Meanwhile, the application value of SPs in vaccine development has attracted increasing attention. This review summarizes the structural characteristics, functional mechanisms, and applications of SPs in recombinant protein production and SP-based vaccines. It also discusses their biological roles, the significance of engineering optimization strategies, and the current challenges, aiming to provide theoretical support and practical guidance for improving recombinant protein yield and advancing SP-based vaccine development. Full article

Small-molecule glucagon-like peptide-1 receptor agonists (GLP-1RAs) represent an innovative advancement in oral therapeutics, addressing key limitations associated with injectable peptide-based incretin therapies. These nonpeptidic agents exert their actions primarily through non-canonical binding orthosteric sites within the GLP-1 receptor transmembrane domain, enabling selective G protein (Gs)-biased signaling with reduced β-arrestin-mediated adverse effects. Orforglipron has notably advanced through Phase 3 clinical development, demonstrating significant reductions in hemoglobin A1c and body weight (up to 7.9%) with favorable tolerability. Conversely, promising candidates such as danuglipron and lotiglipron were discontinued due to hepatotoxicity, underscoring critical safety concerns intrinsic to small-molecule GLP-1RA development. Current clinical candidates, including GSBR-1290, CT-996, and ECC5004, continue to offer substantial potential due to their oral bioavailability, simplified dosing regimens, and favorable gastrointestinal tolerability. Nevertheless, challenges persist regarding hepatic safety, pharmacodynamic variability, and limited long-term outcome data. This review integrates current structural, pharmacological, and clinical evidence, highlights key mechanistic innovations—including biased agonism, covalent binding strategies, and allosteric modulation—and discusses future directions for this rapidly evolving therapeutic class in metabolic disease management. Full article

Heart failure (HF) remains a major global health challenge, driven by multifactorial pathophysiological processes, such as systemic congestion, endothelial dysfunction, and inflammation. While natriuretic peptides are well-established biomarkers for diagnosing and monitoring HF, they do not fully capture the complexity of vascular involvement. CD146, also known as melanoma cell adhesion molecule (MCAM), is a transmembrane glycoprotein primarily expressed on endothelial cells and involved in cell adhesion, vascular permeability, and angiogenesis. Its soluble form (sCD146), released in response to multiple pathophysiological stimuli, including venous and arterial endothelial stretch, oxidative stress, and inflammatory cytokine activation, has emerged as a promising biomarker reflecting both hemodynamic congestion and systemic endothelial stress. This review synthesizes current knowledge on the structure, regulation, and release mechanisms of CD146 and explores its clinical utility in HF. Elevated sCD146 levels have been associated with echocardiographic and radiological indicators of congestion, as well as with adverse outcomes. While promising, its application is limited by variability, lack of standardization, and confounding elevations in non-cardiac conditions, including malignancy. Full article

Background: The PDX2 gene serves as a critical catalytic component in vitamin B6 (VB6) biosynthesis pathways and plays pivotal regulatory roles in plant growth. Methods: To investigate the metabolic regulation of PDX2 (GbPDX2) from Ginkgo biloba in VB6 biosynthesis during kernel development, we successfully cloned this gene and conducted systematic expression profiling through qRT-PCR across multiple tissues and developmental stages. Results: Bioinformatic characterization revealed that GbPDX2 contains a 765-bp coding sequence encoding a 254-amino acid polypeptide. The encoded protein displays typical hydrophilic properties (average hydrophobicity index: −0.32) and was predicted to be an unstable cytosolic protein (instability index: 45.7) lacking signal peptides or transmembrane domains with cytoplasmic localization. Phylogenetic analysis demonstrated that GbPDX2’s closest evolutionary relationship was with its ortholog in Picea sitchensis, which had an amino acid sequence similarity of 83.7% with spruce PsPDX2. Tissue-specific expression analysis revealed a gradient expression profile of Kernel > Exocarp > Leaves > Stems > Roots. The expression level in kernels was significantly higher than that in other tissues (19.7 times that in roots, 8.3 times that in stems, and 5.9 times that in leaves; p < 0.01), with peak transcript levels observed in mature kernels. HPLC quantification established a strong positive correlation between GbPDX2 expression dynamics and VB6 accumulation patterns during kernel maturation (r = 0.92, p < 0.01), and the peak period of VB6 reached 288.9 ± 7.1 μg/g. Conclusions: Our findings provide the first experimental evidence that GbPDX2 spatiotemporally regulates VB6 biosynthesis in ginkgo kernels, offering novel insights into the evolutionary adaptation of vitamin metabolism in gymnosperms. Full article

The mitochondrial phosphate carrier (mPiC), encoded by the nuclear gene SLC25A3, is synthesized with an N-terminus mitochondrial targeting sequence (MTS), enabling its import into the mitochondria. mPiC imports inorganic phosphate (P_i) into the mitochondrial matrix for ATP production and other matrix phosphorylation reactions, as well as regulates mitochondrial Ca²⁺ uptake and buffering of matrix Ca²⁺. PiC also imports copper (Cu), crucial to COX subunit holoenzyme assembly. Variants in SLC25A3 exist and lead to mPiC deficiency (MPCD), cause a rare autosomal recessive disease with no current cure; patients with MPCD usually die within the first year of life. We have developed a novel therapeutic approach using TAT-mPiC fusion protein for cellular delivery since the TAT peptide enables delivery of proteins across biological membranes. We designed, produced, and purified the TAT-mPiC fusion protein. The fusion protein is delivered into the mitochondria and localizes within the mIM, its natural cellular location, as a processed protein. Treatment of mPiC-knockdown cells with TAT-mPiC fusion protein increased cell growth and improved bioenergetic capabilities, as measured by oxygen consumption rate (OCR), ATP production, and reduction in lactate secretion. Most importantly, TAT-mPiC restored P_i and Cu delivery into the mitochondrial matrix. TAT-mPiC fusion protein also restored the mitochondrial activity of cells harboring various mitochondrial defects. This study presents the first successful delivery of a mitochondrial transmembrane carrier using the TAT-fusion system, offering a potential early treatment strategy for newborns with mPiC deficiency. Full article

Auxin response factor (ARF) is a plant-specific transcription factor that responds to changes in auxin levels, regulating various biological processes in plants such as flower development, senescence, lateral root formation, stress response, and secondary metabolite accumulation. In this study, we identified the ARF gene family in Populus euphratica Oliv. using bioinformatics analysis, examining their conserved structural domains, gene structure, expression products, and evolutionary relationships. We found that the 34 PeARF genes were unevenly distributed on 19 chromosomes of P. euphratica. All 56 PeARF proteins were hydrophilic and unstable proteins localized in the nucleus, with secondary structures containing α-helices, extended strands, random coils, and β-turns but lacking transmembrane helices (TM-helices) and signal peptides. Evolutionary analysis divided the PeARF proteins into five subfamilies (A–E), with high conservation observed in the order and number of motifs, domains, gene structure, and other characteristics within each subfamily. Expression pattern analysis revealed that 17 PeARF genes were upregulated during cell growth and heterophylly development. This comprehensive analysis provides insights into the molecular mechanisms of ARF genes in P. euphratica growth, development, and stress response, serving as a basis for further studies on the auxin signaling pathway in P. euphratica. Full article

Lysosomal enzymes are synthesized as N-glycosylated glycoproteins with mannose-6-phosphate (M6P) moieties, which are responsible for their binding to M6P receptors and transporting to the lysosome. In the M6P biosynthetic pathway, a Man₈GlcNAc₂ glycoform is converted to M6P groups through two consecutive enzymatic reactions, including N-acetylglucosamine (GlcNAc)-1-phosphotransferase (GNPT), transferring GlcNAc-1-phosphate from UDP-GlcNAc to the C6 hydroxyl groups of mannose residues, and then, removal of the covering GlcNAc moiety from the GlcNAc-P-mannose phosphodiester was carried out using an α-N-acetylglucosaminidase (referred to as ‘uncovering enzyme’, UCE) in the trans-Golgi network (TGN). Here, we expressed differently tailored versions of the UCE, including four truncated variants, in Escherichia coli. The four variants with the signal peptide, transmembrane domain, propiece and cytoplasmic tail truncated, respectively, were purified by affinity chromatography, and their enzymatic activities were assayed using a UDP-Glo kit. By fusing a maltose-binding protein (MBP) in the N-terminus of the UCE variants, the fusion proteins could be soluble when expressed in E. coli. The highest concentration of the purified enzyme was 80.5 mg/L of fermentation broth. Furthermore, the UCE with the core catalytic domain exhibited the highest uncovering activity. Full article