The Protein Journal最新文献

As a heterogeneous multifactorial disorder, PCOS still has a misty etiology. Its underlying pathophysiological causes can be further elucidated by proteomic analyses and molecular network analysis to understand the interaction pathways involved in the PCOS-associated perturbations. We conducted a proteomic study on ovulatory PCOS serum samples using nano-LCMS/MS technique. Then, we analysed the proteomic profiles of substantially dysregulated proteins by projecting them onto protein interaction mapping and molecular network analysis software tools Gene Mania and STRING. We further investigated the involvement of the affected proteins in different PCOS-associated disorders and classified them through a review of the literature along with functional annotation software tools DAVID and Panther. We found a total of 228 proteins in serum; 109 were found in both ovulatory PCOS and controls, and 42 of those showed a difference of ≥twofold (19 higher in ovulatory PCOS and 23 lower). Among them, 35 proteins exhibited an association with the pathophysiological mechanisms underlying the manifestation of ovulatory PCOS manifestation and their correlations with PCOS-concurrent disorders were revealed. There were also 87 proteins that were only found in ovulatory PCOS and 32 that were only found in controls. We further highlighted significant functional hub molecules within protein interaction networks. Our findings indicated that the ovulatory PCOS involves a wide range of functional molecule derangements, which trigger aberrant biological responses and molecular interactions leading to the emergence of complications associated with ovulatory PCOS. Further omics studies are required to explain the different physiological mechanisms of the functional molecules contributing to the pathogenicity of this heterogeneous syndrome.

Antibiotic resistance presents a major global health threat, especially with ESKAPE pathogens like Serratia marcescens, which exhibit resistance to all known antibiotics. Quorum sensing (QS) is key to its virulence and resistance, emphasizing the need for novel natural antimicrobial agents. This study investigates two plant-derived phenolic compounds, coumaric acid and syringic acid, targeting QS proteins of S. marcescens using in silico molecular docking, molecular dynamics simulations, and in vitro biochemical assays. Validated homology models of eight QS-associated proteins—BsmA, FimA, FimC, FlhD, LuxS, PigP, RsmA, and RssB—were employed for molecular docking studies, ADME (absorption, distribution, metabolism, and excretion) profiling, and 100-ns molecular dynamics (MD) simulations to evaluate ligand-binding stability. Coumaric acid displayed more desirable physicochemical properties (logP 1.79; TPSA 57.53 Ų) compared to syringic acid (logP 1.04; TPSA 75.99 Ų). Binding energy calculations indicated a stronger affinity of coumaric acid for six of the proteins, with the LuxS–coumaric acid complex showing the most significant interaction (ΔGbind − 21.74 ± 3.01 kcal/mol). Analysis of the MD trajectory revealed that coumaric acid enhanced protein stability, as shown by reductions in RMSF (root mean square fluctuation), a more compact Rg (radius of gyration), decreased SASA (solvent-accessible surface area), alterations in the Dictionary of secondary structure of protein (DSSP), and consistent hydrogen bonding. Conversely, syringic acid induced increased conformational flexibility and destabilized alpha-helices and beta-sheets in specific proteins. Principal component analysis (PCA) confirmed tighter conformational clustering in coumaric acid complexes, consistent with improved stabilization. Furthermore, antibacterial assays demonstrated strong inhibitory effects, with MIC values of 700 µg/mL for coumaric acid and 1000 µg/mL for syringic acid. Coumaric acid displayed a bactericidal effect, whereas syringic acid was bacteriostatic. Additionally, time–kill assays revealed a dose-dependent reduction in bacterial growth over 48 h following treatment with varying concentrations of these phenolic acids . Interestingly, qPCR analysis of QS-specific gene expression showed significant downregulation of key QS-regulated genes in response to both compounds, highlighting their potential as quorum-sensing inhibitors and supporting their development as alternative antimicrobial agents against antibiotic-resistant S. marcescens.

Anthrax Toxin Receptor 1 (ANTXR1) is a transmembrane protein involved in various biological processes, including angiogenesis, cell adhesion, and migration. As a receptor for Bacillus anthracis toxins and the oncolytic Seneca Valley virus, ANTXR1 plays pivotal roles in extracellular matrix interactions, actin cytoskeleton organization, and tumor progression. Despite its relevance in cancer biology, ANTXR1 remains understudied from a phosphoproteomics perspective. In this study, we report the phosphoproteomic landscape of the ANTXR1 protein through a unique data integration strategy from a mass spectrometry-based phosphoproteomics perspective. Through robust statistical analyses, conserved phosphorylation events of ANTXR1 across diverse experimental conditions were linked to its upstream kinases and binary interactors to deduce specific events modulated through ANTXR1 phosphorylation. This computational analysis of curated datasets identified conserved ANTXR1 phosphorylation events along with similar and oppositely co-regulated phosphorylation events of adjunct proteins, revealing extensive regulatory networks of ANTXR1. Our findings provide phosphorylation-dependent interaction between ANTXR1 and FLNA and their upstream kinases and phosphobinding motifs, emphasizing their collective role in cell migration. Overall, the study enhances the integrative analysis of mass spectrometry-based phosphoproteomics data through bioinformatics and statistical approaches.

The urease enzyme has an inevitable application in cereal crops, particularly in response to foliar urea application. A holistic and novel approach was employed in the present work with the aim to purify and characterize the wheat leaf urease. This will help in exploring and enhancing its activity in assimilation of foliar urea application and a move towards sustainability. Wheat urease was purified to electrophoretic homogeneity with a 41.98 fold purification and 36.3% recovery. The molecular weight of the native enzyme was found to be ~ 290 kDa by Gel Filtration Chromatography (GFC), and a single band in Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) of ~ 103 kDa deduced its homotrimeric nature. The enzyme had a K_m of 1.0 mM, V_max of 63.25 units mL⁻¹, turnover number of 30.26 min⁻¹, and a specificity constant of 504.33 M⁻¹ sec⁻¹. Further, the optimum pH was 7.5 with 40 °C optimum temperature. The E_a of the purified urease was 61.36 kJ mol⁻¹, with the E_d as 104.3 kJ mol⁻¹. The half-life and D-value decreased with an increase in temperature owing to the rapid loss of its catalytic activity. The z-value was calculated as 44.6 °C. The thermodynamic study revealed the interplay between ΔH, ΔG and ΔS during enzyme deactivation. Histidine was found to be present at the active site and Nickel enhanced the urease activity, whereas copper displayed an inhibitory effect. Hence, this study of wheat urease offers novel insights into an enzyme that has remained largely unexplored despite its inevitable importance in cereal crops. The measures for enhancing its activity in vivo can also be abstracted from this study.

The aim of this study was to design a novel structure-altering polypeptide (SAP) as an anti-microtubule against tumor cells. This series of SAP XA1–XA17 was synthesized by manual solid-phase synthesis and verified by high-performance liquid chromatography (HPLC) and mass spectrometry. Polypeptides were used in three normal cell lines and four tumor cell lines. The optimal polypeptide was selected. Molecular docking of the above optimal polypeptide with tubulin was performed. Tubulin polymerization experiment was performed to investigate effect of optimized peptide to tubulin polymerization. The effect of optimized peptide to cancer in vivo was tested in A549 xenograft tumor mice model. The results of mass spectrometry revealed that the molecular weights of the SAP XA1–XA17 samples were relatively consistent with the theoretical values, whereas the purities of the SAP XA1–XA17 series polypeptide samples were greater than 92.00%. Among the SAP XA1–17 polypeptides, the cell viability kit-8 (CCK-8) assay demonstrated that the XA5 polypeptide was nearly nontoxic to three normal cell lines and had excellent antitumor effects on four cancer cell lines. Molecular docking demonstrated that the polypeptide XA5 preferred tubulin. The docking energies are less than − 5 kcal/mol, verifying the excellent performance of the selected XA5 polypeptide. Tubulin polymerization experiment showed XA5 inhibited tubulin polymerization. In animal study, XA5 administration decreased A549 xenograft tumor weight. The XA5 polypeptide is an effective anti-microtubule drug.

Ataxin-2 (ATXN2), a key RNA-binding protein, regulates RNA metabolism, stress granule formation, and neuronal homeostasis, with dysregulated phosphorylation contributing to Spinocerebellar Ataxia type 2 (SCA2), amyotrophic lateral sclerosis (ALS), and cancer. This review integrates structural biology, phosphoproteomics, and interactome analyses to map six critical phosphosites (S772, T741, S624, S684, S784, S889) within ATXN2’s intrinsically disordered regions. Modulated by kinases GSK3β and CDK13 and phosphatases like INPP5F, these sites orchestrate interactions with RNA-binding partners (e.g., ATXN2L, FXR2, STAU2) and co-regulated proteins (e.g., TP53BP1, NUP153), driving pathogenesis through disrupted autophagy, nucleocytoplasmic transport, and stress granule dynamics. We propose targeted therapies, including GSK3β inhibitors for ALS, antisense oligonucleotides for SCA2, and MTOR modulators for cancer, to restore ATXN2 function. By elucidating phosphocode of ATXN2, this work highlights novel avenues for precision medicine in neurodegenerative and oncogenic diseases.

6% hydroxyethyl starch (HES 130/0.4) is frequently employed to address hypovolemia, ensuring sufficient organ perfusion and oxygen transport. The effects on Apolipoprotein A-I (ApoA-I) were examined at three temperatures—280, 295, and 310 K—through several spectroscopic techniques to explore its possible interaction with the predominant protein in veins. The experimental findings indicated that HES 130/0.4 efficiently extinguished the intrinsic fluorescence of APOA-I. We also assessed the binding sites, binding constant, and thermodynamic parameters, which indicated that HES 130/0.4 can spontaneously associate with APOA-I via hydrogen bonds and van der Waals interactions (ΔG =  − 1.93 × 10⁴ J·mol⁻¹, ΔH =  − 5.63 × 10⁴ J mol⁻¹, and ΔS =  − 119 J mol⁻¹ K⁻¹) with a single binding site and week binding forces (n = 1.03 and K_A = 1.78 × 10³ M⁻¹) at body temperature. Moreover, the structure of APOA-I was significantly altered in the presence of HES 130/0.4. Blood Ca²⁺ and Fe³⁺ will diminish the storage duration. The study provides accurate and thorough foundational data to clarify the binding mechanisms of HES 130/0.4 with APOA-I in vitro, which may help the comprehension of its impact on protein function and toxic mechanism during transit and distribution in the bloodstream.

Nitrate contamination in water sources creates major health risks that primarily affect infants by causing methemoglobinemia (“blue baby syndrome”) while also leading to congenital defects and cancer development. The human body absorbs nitrates mainly through drinking contaminated water. Enzyme nitrate reductase (NR) produced by microorganisms, functions as a key factor in nitrate detoxification. A partial NR gene (GenBank accession: MN833805) from Aspergillus niger PKA16 (KY907172.1) was amplified by employing degenerate primers in this research. The primer sequences were designed based on conserved protein motifs and orthologous diversity analysis of 399 NR protein sequences spanning 127 fungal genera. The NR proteins exhibited an extensive range which demonstrated extensive intra- and interspecies diversity. The multiple conserved domains included nine motifs which remained consistent despite the observed sequence variability. Two highly conserved sequences RLTGKHPFN and PDHGYPLRLV were validated through degenerate-PCR which demonstrated their effectiveness for partial NR gene detection and amplification. In the present study, the developed degenerate primers enable researchers to detect and amplify NR genes from majority of known and unknown fungal strains including those identified through metagenomic studies also. This research establishes fundamental principles for using biotechnology to amplify bioremediatory enzyme nitrate reductase from fungal origin to clean up water and food that contains nitrates, to reduce the risk of ‘blue baby’ disease and cancer.

Bacterial antimicrobial resistance is a great public health threat worldwide, a situation that is much escalated by the rapid propagation of Extended Spectrum β-lactamase (ESBL) enzymes. These can hydrolyze and inactivate a broad range of β-lactams, including third-generation cephalosporins, penicillin, and aztreonam and are known to be associated with various bacterial infections, ranging from uncomplicated urinary tract infections to life-threatening sepsis.Variation is the essential raw material of Darwinian evolution and the accumulation of mutations plays one of the most important roles in it. Sequential acquisition of spontaneous mutations followed by successive rounds of selection can be attributed as one of the major reasons for the rapid diversification of ESBL enzymes. The ESBLs are excellent examples of ‘microevolution’ that led to ‘gain-of-function’ with an extended substrate spectrum. However, acquiring newer phenotypes sometimes comes with fitness costs and different mutational pathways interact with each other, triggering both additive and non-additive fitness to generate a rugged fitness landscape, that influences the path a strain must follow to adapt and evolve under selection pressure. Therefore, it is important to understand the role of mutations in the emergence of these enzyme variants. This review focuses on the understanding of different facades of mutational pathways that lead to the adaptive evolution of ESBL phenotype. The structural and mechanistic basis of the extension of the substrate spectrum by mutations are also discussed.

The bacterial HslVU enzyme complex consists of two components: the HslV protease and the HslU ATPase. This complex share both structural and sequence similarities with the eukaryotic proteasome. HslV becomes functionally active upon engagement with HslU, which inserts its C-terminal helix into a conserved groove within the HslV dimer. This interaction triggers allosteric modulation, thereby initiating HslV’s proteolytic activity. Because the HslVU system is present in pathogenic bacteria but absent in humans, it represents a promising target for antibacterial drug development. This study focuses on the discovery of small molecules that hyperactivate HslV, leading to excessive protein degradation in harmful bacterial strains. By integrating computational modeling with laboratory assays, four triazine-based compounds were identified as potent activators of HslV. These molecules demonstrated high binding affinity in docking simulations, favorable interaction profiles, and significant activation in biochemical assays. Their ED₅₀ values ranged from 0.37 μM to 0.55 μM, indicating strong potency. Furthermore, ADMET evaluations revealed desirable pharmacokinetic and drug-likeness properties. Overall, this work presents effective, non-peptidic small-molecule activators of the HslV protease and provides new insights into chemical modulation of the HslVU system, offering a promising avenue for antibacterial drug discovery.