Biomolecular Concepts最新文献

Tumor necrosis factor receptor-associated factor proteins (TRAFs) are trimeric proteins that play a fundamental role in signaling, acting as intermediaries between the tumor necrosis factor (TNF) receptors and the proteins that transmit the downstream signal. The monomeric subunits of all the TRAF family members share a common tridimensional structure: a C-terminal globular domain and a long coiled-coil tail characterizing the N-terminal section. In this study, the dependence of the TRAF2 dynamics on the length of its tail was analyzed in silico. In particular, we used the available crystallographic structure of a C-terminal fragment of TRAF2 (168 out of 501 a.a.), TRAF2-C, and that of a longer construct, addressed as TRAF2-plus, that we have re-constructed using the AlphaFold2 code. The results indicate that the longer N-terminal tail of TRAF2-plus has a strong influence on the dynamics of the globular regions in the protein C-terminal head. In fact, the quaternary interactions among the TRAF2-C subunits change asymmetrically in time, while the movements of TRAF2-plus monomers are rather limited and more ordered than those of the shorter construct. Such findings shed a new light on the dynamics of TRAF subunits and on the protein mechanism in vivo, since TRAF monomer-trimer equilibrium is crucial for several reasons (receptor recognition, membrane binding, hetero-oligomerization).

Human endo-lysosomes possess a class of proteins called TPC channels on their membrane, which are essential for proper cell functioning. This protein family can be functionally studied by expressing them in plant vacuoles. Inhibition of hTPC activity by naringenin, one of the main flavonoids present in the human diet, has the potential to be beneficial in severe human diseases such as solid tumor development, melanoma, and viral infections. We attempted to identify the molecular basis of the interaction between hTPC2 and naringenin, using ensemble docking on molecular dynamics (MD) trajectories, but the specific binding site remains elusive, posing a challenge that could potentially be addressed in the future by increased computational power in MD and the combined use of microscopy techniques such as cryo-EM.

The study of the interaction between lipid membranes and amyloidogenic peptides is a turning point for understanding the processes involving the cytotoxicity of peptides involved in neurodegenerative diseases. In this work, we perform an experimental study of model membrane-lysozyme interaction to understand how the formation of amyloid fibrils can be affected by the presence of polar and zwitterionic phospholipid molecules (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine [POPC] and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol [POPG]). The study was conducted above and below the critical micellar concentration (CMC) using dynamic light scattering (DLS), atomic force microscopy (AFM), UV-Vis spectrophotometry, and the quartz crystal microbalance (QCM). Our results show that the presence of phospholipids appears to be a factor favoring the formation of amyloid aggregates. Spectrophotometric and DLS data revealed that the quantity of $\beta$ -structure increases in the presence of POPG and POPC at different concentrations. The presence of POPG and POPC increases the speed of the nucleation process, without altering the overall structures of the fibrillar final products.

Investigating biological mechanisms in ever greater detail requires continuous advances in microscopy techniques and setups. Total internal reflection fluorescence (TIRF) microscopy is a well-established technique for visualizing processes on the cell membrane. TIRF allows studies down to the single molecule level, mainly in single-colour applications. Instead, multicolour setups are still limited. Here, we describe our strategies for implementing a multi-channel TIRF microscopy system capable of simultaneous two-channel excitation and detection, starting from a single-colour commercial setup. First, we report some applications at high molecule density and then focus on the challenges we faced for achieving the single molecule level simultaneously in different channels, showing that rigorous optimizations on the setup are needed to increase its sensitivity up to this point, from camera setting to background minimization. We also discuss our strategies regarding crucial points of fluorescent labelling for this type of experiment: labelling strategy, kind of probe, efficiency, and orthogonality of the reaction, all of which are aspects that can influence the achievable results. This work may provide useful guidelines for setting up advanced single-molecule multi-channel TIRF experiments to obtain insights into interaction mechanisms on the cell membrane of living cells.

Vaccination through the upper respiratory tract is a promising strategy, and particulate antigens, such as antigens associated with nanoparticles, triggered a stronger immune response than the sole antigens. Cationic maltodextrin-based nanoparticles loaded with phosphatidylglycerol (NPPG) are efficient for intranasal vaccination but non-specific to trigger immune cells. Here we focused on phosphatidylserine (PS) receptors, specifically expressed by immune cells including macrophages, to improve nanoparticle targeting through an efferocytosis-like mechanism. Consequently, the lipids associated with NPPG have been substituted by PS to generate cationic maltodextrin-based nanoparticles with dipalmitoyl-phosphatidylserine (NPPS). Both NPPS and NPPG exhibited similar physical characteristics and intracellular distribution in THP-1 macrophages. NPPS cell entry was faster and higher (two times more) than NPPG. Surprisingly, competition of PS receptors with phospho-L-serine did not alter NPPS cell entry and annexin V did not preferentially interact with NPPS. Although the protein association is similar, NPPS delivered more proteins than NPPG in cells. On the contrary, the proportion of mobile nanoparticles (50%), the movement speed of nanoparticles (3 µm/5 min), and protein degradation kinetics in THP-1 were not affected by lipid substitution. Together, the results indicate that NPPS enter cells and deliver protein better than NPPG, suggesting that modification of the lipids of cationic maltodextrin-based nanoparticles may be a useful strategy to enhance nanoparticle efficacy for mucosal vaccination.

Diabetes is accompanied by inflammation and oxidation. Supplementation of anti-inflammatory and antioxidant compounds can prevent the progression of diabetes. This study aimed to investigate the effects of supplementation of Nannochloropsis oculata microalgae (NOM) on the inflammatory and antioxidant responses in diabetic rats. Sixty male rats were divided into six groups as diabetic and non-diabetic rats receiving 0, 10 and 20 mg/kg of body weight of NOM daily for 21 days. Body weight, the serum concentrations of insulin and glucose and the tissue concentrations of interleukin-1β (IL-1β), tumor necrosis factor-alpha (TNF-α), nuclear factor kappa B (NF-κB), interleukin-6 (IL-6), malondialdehyde (MDA), ferric reducing antioxidant power (FRAP), superoxide dismutase (SOD), glutathione peroxidase (GPx) were assessed. The results showed that induction of diabetes significantly reduced the body weight, the serum concentrations of insulin and the tissue concentrations of SOD, FRAP and GPx while increasing the concentrations of glucose, MDA, IL-1β, IL-6, NF-κB and TNF-α. Daily oral administration of NOM (10 and 20 mg/kg) significantly maintained the body weight, the serum concentrations of insulin and the tissue concentrations of SOD, FRAP and GPx while preventing the increase in the concentrations of glucose, MDA, IL-1β and TNF-α. In conclusion, diabetes caused inflammation and oxidation while NOM worked as a natural anti-inflammatory and antioxidant compound.

Growth factors and cytokines activate signal transduction pathways and regulate gene expression in eukaryotes. Intracellular domains of activated receptors recruit several protein kinases as well as transcription factors that serve as platforms or hubs for the assembly of multi-protein complexes. The signaling hubs involved in a related biologic function often share common interaction proteins and target genes. This functional connectivity suggests that a pairwise comparison of protein interaction partners of signaling hubs and network analysis of common partners and their expression analysis might lead to the identification of critical nodes in cellular signaling. A pairwise comparison of signaling hubs across several related pathways might reveal novel signaling modules. Analysis of protein interaction connectome by Venn (PIC-Venn) of transcription factors STAT1, STAT3, NFKB1, RELA, FOS, and JUN, and their common interaction network suggested that BRCA1 and TSC22D3 function as critical nodes in immune responses by connecting the signaling hubs into signaling modules. Transcriptional regulation of critical hubs may play a major role in the lung epithelial cells in response to SARS-CoV-2 and in COVID-19 patients. Mutations and differential expression levels of these critical nodes and modules in pathological conditions might deregulate signaling pathways and their target genes involved in inflammation. Biological connectivity emerges from the structural connectivity of interaction networks across several signaling hubs in related pathways. The main objectives of this study are to identify critical hubs, critical nodes, and modules involved in the signal transduction pathways of innate and adaptive immunity. Application of PIC-Venn to several signaling hubs might reveal novel nodes and modules that can be targeted by small regulatory molecules to simultaneously activate or inhibit cell signaling in health and disease.

Mr4511 from Methylobacterium radiotolerans is a photoreceptor of the light, oxygen voltage (LOV) family, binding flavin mononucleotide (FMN) as a chromophore. It exhibits the prototypical LOV photocycle, with the reversible formation of an FMN-Cys71 adduct via fast decay of the FMN triplet state. Mr4511 has high potential as a photosensitiser for singlet oxygen (SO) upon mutation of C71. Mr4511-C71S shows a triplet lifetime (τ _T) of several hundreds of microseconds, ensuring efficient energy transfer to dioxygen to form SO. In this work, we have explored the potential diffusion pathways for dioxygen within Mr4511 using molecular dynamics (MD) simulations. The structural model of wild-type (wt) Mr4511 showed a dimeric structure stabilised by a strong leucine zipper at the two C-terminal helical ends. We then introduced in silico the C71S mutation and analysed transient and persistent oxygen channels. MD simulations indicate that the chromophore binding site is highly accessible to dioxygen. Mutations that might favour SO generation were designed based on their position with respect to FMN and the oxygen channels. In particular, the C71S-Y61T and C71S-Y61S variants showed an increased diffusion and persistence of oxygen molecules inside the binding cavity.

Calcium signaling controls a large variety of cell functions, including proliferation and apoptosis, and plays a major role in neoplastic transformation. Prostate cancer (PCa) is one of the most common malignancies in men. The transition to castration-resistant prostate cancer (CRPC), a lethal form that is still lacking an effective cure, could be influenced by fine tuning intracellular calcium ([Ca²⁺]_i) homeostasis. This study investigates [Ca²⁺]_i dynamics in metastatic PCa cell lines that mimic the progression of PCa to CRPC: (i) well differentiated LNCaP cells that require androgen for survival, and (ii) poorly differentiated, highly aggressive androgen-insensitive prostate cancer (AIPC) PC3 and DU145 cells. In AIPC cells, ATP induces a fast rise in [Ca²⁺]_i, due to release from intracellular stores and sensitive to phospholipase C inhibitors, while LNCaP cells do not respond to ATP challenge. Moreover, AIPC cells showed a reduced capacity to store Ca²⁺ in thapsigargin-sensitive stores and limited store-operated calcium entry, with respect to androgen-dependent LNCaP cells. Finally, green tea extract causes [Ca²⁺]_i elevation and inhibits proliferation in PC3 and DU145 cells, but is ineffective in LNCaP cells. The consequences of these differences are discussed and interpreted in this study with reference to previously proposed models for Ca²⁺ dependence of prostate carcinogenesis.

Extracellular vesicles (EVs)-mediated communication relies not only on the delivery of complex molecular cargoes as lipids, proteins, genetic material, and metabolites to their target cells but also on the modification of the cell surface local properties induced by the eventual fusion of EVs' membranes with the cells' plasma membrane. Here we applied scanning calorimetry to study the phase transition of single phospholipid (DMPC) monolamellar vesicles, investigating the thermodynamical effects caused by the fusion of doping amounts of mesenchymal stem cells-derived EVs. Specifically, we studied EVs-induced consequences on the lipids distributed in the differently curved membrane leaflets, having different density and order. The effect of EV components was found to be not homogeneous in the two leaflets, the inner (more disordered one) being mainly affected. Fusion resulted in phospholipid membrane flattening associated with lipid ordering, while the transition cooperativity, linked to membrane domains' coexistence during the transition process, was decreased. Our results open new horizons for the investigation of the peculiar effects of EVs of different origins on target cell membrane properties and functionality.