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International Journal of Molecular... Jun 2024Within the sequence of the gene, more than 50 polymorphisms, resulting from single-nucleotide polymorphisms (SNPs), have been described. Some of them play an important...
Within the sequence of the gene, more than 50 polymorphisms, resulting from single-nucleotide polymorphisms (SNPs), have been described. Some of them play an important role as specific genetic markers in the process of carcinogenesis and for therapeutic purposes. In this publication, we present methods we have developed that enable the specific and unambiguous identification of four polymorphisms that result in amino acid changes: c. 142C > G, c. 355G > T, c. 1294C > G, and c. 1358A > G. Our studies are based on cleaved amplified polymorphic sequences (CAPSs) and artificially created restriction site (ACRS) PCR techniques; therefore, they require only basic laboratory equipment and low financial outlays. Utilizing the described methods allows for the reduction of research time and cost, and the minimization of errors. Their effectiveness and efficiency depend on the careful design of appropriate primers and the precise selection of suitable restriction enzymes. As a result, further confirmation by sequencing is not necessary. Using the developed method, we examined 63 patients diagnosed with lung cancer and observed a 1.5 to 2.1 times higher frequency of the analyzed single-nucleotide polymorphisms compared to the frequency in the European population.
Topics: Humans; Cytochrome P-450 CYP1B1; Lung Neoplasms; Polymorphism, Single Nucleotide; Polymerase Chain Reaction; Female; Male; Middle Aged; Aged
PubMed: 38928381
DOI: 10.3390/ijms25126676 -
International Journal of Molecular... Jun 2024Integrin αβ mediates platelet aggregation by binding the Arginyl-Glycyl-Aspartic acid (RGD) sequence of fibrinogen. RGD binding occurs at a site topographically...
Integrin αβ mediates platelet aggregation by binding the Arginyl-Glycyl-Aspartic acid (RGD) sequence of fibrinogen. RGD binding occurs at a site topographically proximal to the α and β subunits, promoting the conformational activation of the receptor from bent to extended states. While several experimental approaches have characterized RGD binding to αβ integrin, applying computational methods has been significantly more challenging due to limited sampling and the need for a priori information regarding the interactions between the RGD peptide and integrin. In this study, we employed all-atom simulations using funnel metadynamics (FM) to evaluate the interactions of an RGD peptide with the α and β subunits of integrin. FM incorporates an external history-dependent potential on selected degrees of freedom while applying a funnel-shaped restraint potential to limit RGD exploration of the unbound state. Furthermore, it does not require a priori information about the interactions, enhancing the sampling at a low computational cost. Our FM simulations reveal significant molecular changes in the β subunit of integrin upon RGD binding and provide a free-energy landscape with a low-energy binding mode surrounded by higher-energy prebinding states. The strong agreement between previous experimental and computational data and our results highlights the reliability of FM as a method for studying dynamic interactions of complex systems such as integrin.
Topics: Oligopeptides; Platelet Glycoprotein GPIIb-IIIa Complex; Protein Binding; Humans; Molecular Dynamics Simulation; Blood Platelets; Binding Sites; Integrin beta3
PubMed: 38928286
DOI: 10.3390/ijms25126580 -
International Journal of Molecular... Jun 2024The construction of peptides to mimic heterogeneous proteins such as type I collagen plays a pivotal role in deciphering their function and pathogenesis. However,...
The construction of peptides to mimic heterogeneous proteins such as type I collagen plays a pivotal role in deciphering their function and pathogenesis. However, progress in the field has been severely hampered by the lack of capability to create stable heterotrimers with desired functional sequences and without the effect of homotrimers. We have herein developed a set of triblock peptides that can assemble into collagen mimetic heterotrimers with desired amino acids and are free from the interference of homotrimers. The triblock peptides comprise a central collagen-like block and two oppositely charged N-/C-terminal blocks, which display inherent incompetency of homotrimer formation. The favorable electrostatic attraction between two paired triblock peptides with complementary terminal charged sequences promptly leads to stable heterotrimers with controlled chain composition. The independence of the collagen-like block from the two terminal blocks endows this system with the adaptability to incorporate desired amino acid sequences while maintaining the heterotrimer structure. The triblock peptides provide a versatile and robust tool to mimic the composition and function of heterotrimer collagen and may have great potential in the design of innovative peptides mimicking heterogeneous proteins.
Topics: Peptides; Collagen; Protein Multimerization; Amino Acid Sequence; Collagen Type I; Static Electricity
PubMed: 38928256
DOI: 10.3390/ijms25126550 -
International Journal of Molecular... Jun 2024Mcl-1 (myeloid cell leukemia 1), a member of the Bcl-2 family, is upregulated in various types of cancer. Peptides representing the BH3 (Bcl-2 homology 3) region of...
Mcl-1 (myeloid cell leukemia 1), a member of the Bcl-2 family, is upregulated in various types of cancer. Peptides representing the BH3 (Bcl-2 homology 3) region of pro-apoptotic proteins have been demonstrated to bind the hydrophobic groove of anti-apoptotic Mcl-1, and this interaction is responsible for regulating apoptosis. Structural studies have shown that, while there is high overall structural conservation among the anti-apoptotic Bcl-2 (B-cell lymphoma 2) proteins, differences in the surface groove of these proteins facilitates binding specificity. This binding specificity is crucial for the mechanism of action of the Bcl-2 family in regulating apoptosis. Bim-based peptides bind specifically to the hydrophobic groove of Mcl-1, emphasizing the importance of these interactions in the regulation of cell death. Molecular docking was performed with BH3-like peptides derived from Bim to identify high affinity peptides that bind to Mcl-1 and to understand the molecular mechanism of their interactions. The interactions of three identified peptides, E2gY, E2gI, and XXA1_F3dI, were further evaluated using 250 ns molecular dynamics simulations. Conserved hydrophobic residues of the peptides play an important role in their binding and the structural stability of the complexes. Understanding the molecular basis of interaction of these peptides will assist in the development of more effective Mcl-1 specific inhibitors.
Topics: Myeloid Cell Leukemia Sequence 1 Protein; Humans; Protein Binding; Molecular Docking Simulation; Peptides; Molecular Dynamics Simulation; Antineoplastic Agents; Hydrophobic and Hydrophilic Interactions; Binding Sites; Amino Acid Sequence; Bcl-2-Like Protein 11
PubMed: 38928234
DOI: 10.3390/ijms25126529 -
International Journal of Molecular... Jun 2024Pollen from common ragweed is an important allergen source worldwide and especially in western and southern Romania. More than 100 million patients suffer from symptoms...
Pollen from common ragweed is an important allergen source worldwide and especially in western and southern Romania. More than 100 million patients suffer from symptoms of respiratory allergy (e.g., rhinitis, asthma) to ragweed pollen. Among the eleven characterized allergens, Amb a 6 is a non-specific lipid transfer protein (nsLTP). nsLTPs are structurally stable proteins in pollen and food from different unrelated plants capable of inducing severe reactions. The goal of this study was to produce Amb a 6 as a recombinant and structurally folded protein (rAmb a 6) and to characterize its physicochemical and immunological features. rAmb a 6 was expressed in cells as a secreted protein and characterized by mass spectrometry and circular dichroism (CD) spectroscopy regarding molecular mass and fold, respectively. The IgE-binding frequency towards the purified protein was evaluated using sera from 150 clinically well-characterized ragweed-allergic patients. The allergenic activities of rAmb a 6 and the nsLTP from the weed (Par j 2) were evaluated in basophil activation assays. rAmb a 6-specific IgE reactivity was associated with clinical features. Pure rAmb a 6 was obtained by insect cell expression. Its deduced molecular weight corresponded to that determined by mass spectrometry (i.e., 10,963 Da). rAmb a 6 formed oligomers as determined by SDS-PAGE under non-reducing conditions. According to multiple sequence comparisons, Amb a 6 was a distinct nsLTP with less than 40% sequence identity to currently known plant nsLTP allergens, except for nsLTP from (i.e., 52%). rAmb a 6 is an important ragweed allergen recognized by 30% of ragweed pollen allergic patients. For certain patients, rAmb a 6-specific IgE levels were higher than those specific for the major ragweed allergen Amb a 1 and analysis also showed a higher allergenic activity in the basophil activation test. rAmb a 6-positive patients suffered mainly from respiratory symptoms. The assumption that Amb a 6 is a source-specific ragweed allergen is supported by the finding that none of the patients showing rAmb a 6-induced basophil activation reacted with Par j 2 and only one rAmb a 6-sensitized patient had a history of plant food allergy. Immunization of rabbits with rAmb a 6 induced IgG antibodies which strongly inhibited IgE binding to rAmb a 6. Our results demonstrate that Amb a 6 is an important source-specific ragweed pollen allergen that should be considered for diagnosis and allergen-specific immunotherapy of ragweed pollen allergy.
Topics: Humans; Allergens; Immunoglobulin E; Antigens, Plant; Animals; Carrier Proteins; Plant Proteins; Female; Rhinitis, Allergic, Seasonal; Male; Adult; Ambrosia; Spodoptera; Recombinant Proteins; Amino Acid Sequence; Sf9 Cells; Middle Aged; Plant Extracts
PubMed: 38928218
DOI: 10.3390/ijms25126513 -
International Journal of Molecular... Jun 2024Paraformaldehyde (PFA) fixation is the preferred method for preserving tissue architecture for anatomical and pathological observations. Meanwhile, PFA reacts with the...
Paraformaldehyde (PFA) fixation is the preferred method for preserving tissue architecture for anatomical and pathological observations. Meanwhile, PFA reacts with the amine groups of biomolecules to form chemical cross-linking, which preserves RNA within the tissue. This has great prospects for RNA sequencing to characterize the molecular underpinnings after anatomical and pathological observations. However, RNA is inaccessible due to cross-linked adducts forming between RNA and other biomolecules in prolonged PFA-fixed tissue. It is also difficult to perform reverse transcription and PCR, resulting in low sequencing sensitivity and reduced reproducibility. Here, we developed a method to perform RNA sequencing in PFA-fixed tissue, which is easy to use, cost-effective, and allows efficient sample multiplexing. We employ cross-link reversal to recover RNA and library construction using random primers without artificial fragmentation. The yield and quality of recovered RNA significantly increased through our method, and sequencing quality metrics and detected genes did not show any major differences compared with matched fresh samples. Moreover, we applied our method for gene expression analysis in different regions of the mouse brain and identified unique gene expression profiles with varied functional implications. We also find significant dysregulation of genes involved in Alzheimer's disease (AD) pathogenesis within the medial septum (MS)/vertical diagonal band of Broca (VDB) of the 5×FAD mouse brain. Our method can thus increase the performance of high-throughput RNA sequencing with PFA-fixed samples and allows longitudinal studies of small tissue regions isolated by their in situ context.
Topics: Formaldehyde; Animals; Mice; Brain; Tissue Fixation; Sequence Analysis, RNA; Alzheimer Disease; Polymers; Gene Expression Profiling; High-Throughput Nucleotide Sequencing; RNA
PubMed: 38928210
DOI: 10.3390/ijms25126504 -
International Journal of Molecular... Jun 2024Complex gut microbiota increases chickens' resistance to enteric pathogens. However, the principles of this phenomenon are not understood in detail. One of the...
Complex gut microbiota increases chickens' resistance to enteric pathogens. However, the principles of this phenomenon are not understood in detail. One of the possibilities for how to decipher the role of gut microbiota in chickens' resistance to enteric pathogens is to systematically characterise the gene expression of individual gut microbiota members colonising the chicken caecum. To reach this aim, newly hatched chicks were inoculated with bacterial species whose whole genomic sequence was known. Total protein purified from the chicken caecum was analysed by mass spectrometry, and the obtained spectra were searched against strain-specific protein databases generated from known genomic sequences. , sp. and did not utilise carbohydrates when colonising the chicken caecum. On the other hand, , , , , , , , and fermented carbohydrates. was the only motile bacterium, and expressed the type VI secretion system. Classification of in vivo expression is key for understanding the role of individual species in complex microbial populations colonising the intestinal tract. Knowledge of the expression of motility, the type VI secretion system, and preference for carbohydrate or amino acid fermentation is important for the selection of bacteria for defined competitive exclusion products.
Topics: Animals; Chickens; Gastrointestinal Microbiome; Amino Acids; Type VI Secretion Systems; Carbohydrate Metabolism; Cecum; Bacteria
PubMed: 38928209
DOI: 10.3390/ijms25126505 -
International Journal of Molecular... Jun 2024Arc (also known as Arg3.1) is an activity-dependent immediate early gene product enriched in neuronal dendrites. Arc plays essential roles in long-term potentiation,...
Arc (also known as Arg3.1) is an activity-dependent immediate early gene product enriched in neuronal dendrites. Arc plays essential roles in long-term potentiation, long-term depression, and synaptic scaling. Although its mechanisms of action in these forms of synaptic plasticity are not completely well established, the activities of Arc include the remodeling of the actin cytoskeleton, the facilitation of AMPA receptor (AMPAR) endocytosis, and the regulation of the transcription of AMPAR subunits. In addition, Arc has sequence and structural similarity to retroviral Gag proteins and self-associates into virus-like particles that encapsulate mRNA and perhaps other cargo for intercellular transport. Each of these activities is likely to be influenced by Arc's reversible self-association into multiple oligomeric species. Here, we used mass photometry to show that Arc exists predominantly as monomers, dimers, and trimers at approximately 20 nM concentration in vitro. Fluorescence fluctuation spectroscopy revealed that Arc is almost exclusively present as low-order (monomer to tetramer) oligomers in the cytoplasm of living cells, over a 200 nM to 5 μM concentration range. We also confirmed that an α-helical segment in the N-terminal domain contains essential determinants of Arc's self-association.
Topics: Protein Multimerization; Humans; Cytoskeletal Proteins; Nerve Tissue Proteins; Animals
PubMed: 38928159
DOI: 10.3390/ijms25126454 -
International Journal of Molecular... Jun 2024is the dominant parasitic natural enemy of aphids. Elucidating the molecular mechanism of host recognition of would improve its biological control effect. Chemosensory...
is the dominant parasitic natural enemy of aphids. Elucidating the molecular mechanism of host recognition of would improve its biological control effect. Chemosensory proteins (CSPs) play a crucial role in insect olfactory systems and are mainly involved in host localization. In this study, a total of nine CSPs of with complete open reading frames were identified based on antennal transcriptome data. Phylogenetic analysis revealed that AgifCSPs were mainly clustered into three subgroups (AgifCSP1/2/7/8, AgifCSP3/9, and AgifCSP4/5/6). showed high expression in the antennae of both sexes. Moreover, was found to be specifically expressed in the antennae. In addition, fluorescent binding assays revealed that AifCSP5 had greater affinities for 7 of 32 volatile odor molecules from various sources. Molecular docking and site-directed mutagenesis results revealed that the residue at which AgifCSP5 binds to these seven plant volatiles is Tyr75. Behavior tests further confirmed that -2-nonenal, one of the seven active volatiles in the ligand binding test, significantly attracted female adults at a relatively low concentration of 10 mg/mL. In conclusion, AgifCSP5 may be involved in locating aphid-infested crops from long distances by detecting and binding -2-nonenal. These findings provide a theoretical foundation for further understanding the olfactory recognition mechanisms and indirect aphid localization behavior of from long distances by first identifying the host plant of aphids.
Topics: Animals; Aphids; Insect Proteins; Phylogeny; Female; Male; Host-Parasite Interactions; Arthropod Antennae; Molecular Docking Simulation; Amino Acid Sequence; Receptors, Odorant; Wasps
PubMed: 38928098
DOI: 10.3390/ijms25126392 -
International Journal of Molecular... Jun 2024The downstream receptor kinase (Drk), a homologue of human GRB2, participates in the signal transduction from the extracellular to the intracellular environment. Drk...
The downstream receptor kinase (Drk), a homologue of human GRB2, participates in the signal transduction from the extracellular to the intracellular environment. Drk receives signals through the interaction of its Src homology 2 (SH2) domain with the phosphorylated tyrosine residue in the receptor tyrosine kinases (RTKs). Here, we present the solution NMR structure of the SH2 domain of Drk (Drk-SH2), which was determined in the presence of a phosphotyrosine (pY)-containing peptide derived from a receptor tyrosine kinase, Sevenless (Sev). The solution structure of Drk-SH2 possess a common SH2 domain architecture, consisting of three β strands imposed between two α helices. Additionally, we interpret the site-specific interactions of the Drk-SH2 domain with the pY-containing peptide through NMR titration experiments. The dynamics of Drk-SH2 were also analysed through NMR-relaxation experiments as well as the molecular dynamic simulation. The docking simulations of the pY-containing peptide onto the protein surface of Drk-SH2 provided the orientation of the peptide, which showed a good agreement with the analysis of the SH2 domain of GRB2.
Topics: src Homology Domains; Drosophila Proteins; Molecular Dynamics Simulation; Protein Binding; Animals; Humans; Receptor Protein-Tyrosine Kinases; GRB2 Adaptor Protein; Molecular Docking Simulation; Binding Sites; Amino Acid Sequence; Magnetic Resonance Spectroscopy
PubMed: 38928093
DOI: 10.3390/ijms25126386