Molecular BioSystems最新文献

The recognition and association between the Ca²⁺/calmodulin-activated protein kinase II-α (CaMKIIα) and the multi-PDZ domain protein 1 (MUPP1) plays an important role in the sperm acrosome reaction and human fertilization. Previously, we have demonstrated that the MUPP1 PDZ11 domain is the primary binding partner of the CaMKIIα C-terminal tail, which can be targeted by a rationally designed sia peptide with nanomolar affinity. Here, we further introduced an orthogonal noncovalent interaction (ONI) system between a native hydrogen bond and a designed halogen bond across the complex interface of the PDZ11 domain with the sia [Asn-1Phe] peptide mutant, where the halogen bond was formed by substituting the o-hydrogen atom of the benzene ring of the peptide Phe-1 residue with a halogen atom (F, Cl, Br or I). Molecular dynamics simulations and high-level theoretical calculations suggested that bromine (Br) is a good compromise between the halogen-bonding strength and steric hindrance effect due to introduction of a bulkier halogen atom into the tightly packed complex interface. Fluorescence spectroscopy assays revealed that the resulting o-Br-substituted peptide (K_d = 18 nM) exhibited an ～7.6-fold affinity increase relative to its native counterpart (K_d = 137 nM). In contrast, the p-Br-substituted peptide, a negative control that is unable to establish the ONI according to structure-based analysis, has decreased affinity (K_d = 210 nM) upon halogenation.

Several developments regarding the analysis of gene co-expression profiles using complex network theory have been reported recently. Such approaches usually start with the construction of an unweighted gene co-expression network, therefore requiring the selection of a suitable threshold defining which pairs of vertices will be connected. We aimed at addressing such an important problem by suggesting and comparing five different approaches for threshold selection. Each of the methods considers a respective biologically-motivated criterion for electing a potentially suitable threshold. A set of 21 microarray experiments from different biological groups was used to investigate the effect of applying the five proposed criteria to several biological situations. For each experiment, we used the Pearson correlation coefficient to measure the relationship between each gene pair, and the resulting weight matrices were thresholded considering several values, generating respective adjacency matrices (co-expression networks). Each of the five proposed criteria was then applied in order to select the respective threshold value. The effects of these thresholding approaches on the topology of the resulting networks were compared by using several measurements, and we verified that, depending on the database, the impact on the topological properties can be large. However, a group of databases was verified to be similarly affected by most of the considered criteria. Based on such results, it can be suggested that when the generated networks present similar measurements, the thresholding method can be chosen with greater freedom. If the generated networks are markedly different, the thresholding method that better suits the interests of each specific research study represents a reasonable choice.

Mass spectrometry imaging (MSI) of animal tissues has become an important tool for in situ molecular analyses and biomarker studies in several clinical areas, but there are few applications in parasitological studies. Leishmaniasis is a neglected tropical disease, and experimental mouse models have been essential to evaluate pathological and immunological processes and to develop diagnostic methods. Herein we have employed MALDI MSI to examine peptides and low molecular weight proteins (2 to 20 kDa) differentially expressed in the liver during visceral leishmaniasis in mice models. We analyzed liver sections of Balb/c mice infected with Leishmania infantum using the SCiLS Lab software for statistical analysis, which facilitated data interpretation and thus highlighted several key proteins and/or peptides. We proposed a decision tree classification for visceral leishmaniasis with distinct phases of the disease, which are named here as healthy, acute infection and chronic infection. Among others, the ion of m/z 4963 was the most important to identify acute infection and was tentatively identified as Thymosin β4. This peptide was previously established as a recovery factor in the human liver and might participate in the response of mice to Leishmania infection. This preliminary investigation shows the potential of MALDI MSI to complement classical compound selective imaging techniques and to explore new features not yet recognized by these approaches.

Irreversible denaturation of membrane proteins in detergent solutions is similar to unfolding of water-soluble multidomain proteins and represents a complex, multistage process. Pore-forming proteins of Gram-negative bacteria are heat-modifiable proteins, i.e., proteins altering their molecular forms (trimers or monomers), and accordingly, their electrophoretic mobilities depending upon denaturation conditions. There are still some contradictory data on the peculiarities of the conformational changes in the porin structure with temperature. Some authors demonstrated the loss of the porin trimeric structure only after unfolding of monomer subunits. Other researchers initially observed the dissociation of porin oligomers into the folded monomers. Using SDS-PAGE, spectroscopic methods and differential scanning calorimetry, a detailed study of thermally induced changes in the spatial structure of OmpF porin from the fish pathogen Yersinia ruckeri (Yr-OmpF) was carried out. The data obtained allowed us to conclude unambiguously that changes in the spatial structure of the monomers of Yr-OmpF precede the dissociation of the porin trimer.

The emergence of new protein-coding genes in a specific lineage or species provides raw materials for evolutionary adaptations. Until recently, the biology of new genes emerging particularly from non-genic sequences remained unexplored. Although the new genes are subjected to variable selection pressure and face rapid deletion, some of them become functional and are retained in the gene pool. To acquire functional novelties, new genes often get integrated into the pre-existing ancestral networks. However, the mechanism by which young proteins acquire novel interactions remains unanswered till date. Since structural orientation contributes hugely to the mode of proteins' physical interactions, in this regard, we put forward an interesting question – Do new genes encode proteins with stable folds? Addressing the question, we demonstrated that the intrinsic disorder inversely correlates with the evolutionary gene ages – i.e. young proteins are richer in intrinsic disorder than the ancient ones. We further noted that young proteins, which are initially poorly connected hubs, prefer to be structurally more disordered than well-connected ancient proteins. The phenomenon strikingly defies the usual trend of well-connected proteins being highly disordered in structure. We justified that structural disorder might help poorly connected young proteins to undergo promiscuous interactions, which provides the foundation for novel protein interactions. The study focuses on the evolutionary perspectives of young proteins in the light of structural adaptations.

The multidrug resistance (MDR) system actively pumps antibiotics out of cells causing serious health problems. During the pumping, AcrB (one of the key components of MDR) undergoes a series of large-scale and proton-motive conformational changes. Capturing the conformational changes through all-atom simulations is challenging. Here, we implement a hybrid coarse-grained force field to investigate the conformational changes of AcrB in the porter domain under different protonation states of Asp407/Asp408 in the trans-membrane domain. Our results show that protonation of Asp408 in monomer III (extrusion) stabilizes the asymmetric structure of AcrB; deprotonation of Asp408 induces clear opening of the entrance and closing of the exit leading to the transition from extrusion to access state. The structural changes in the porter domain of AcrB are strongly coupled with the proton translocation stoichiometry in the trans-membrane domain. Moreover, our simulations support the postulation that AcrB should adopt the symmetric resting state in a substrate-free situation.

Giardia intestinalis is an intestinal parasite that causes diarrhea in humans and animals worldwide. The enolase of G. intestinalis (GiENO) participates in its glycolysis pathway and is abundantly expressed in the parasite cytosol; however, its localization on the surface of trophozoites and cysts has been demonstrated. Enolases from bacteria and parasites can have different functions and are considered moonlighting proteins, for example, as a cell surface plasminogen receptor. In relation to GiENO, no studies have been performed about its possible participation as a plasminogen receptor. In this work, we employed molecular docking and multiscale molecular dynamics (MD) simulations to explore the possible interactions of human plasminogen (HsPLG) with the open and closed GiENO conformations. Our proposed GiENO plasminogen binding site (PLGBs) was identified at Lys266 based on the sequence comparison with bacterial enolase known to act as a plasminogen receptor. Our docking results performed with multiple MD snapshots of the closed GiENO conformation showed that Lys266 preferentially binds to the K5 domain of HsPLG. On the other hand, open GiENO conformations from all-atom and coarse-grained simulations indicated a high preference of the HsPLG K4 domain for lysine residues 186 and 188. Furthermore, we identified a potential N-glycosylation site of GiENO which suggests a possible explanation for the parasite cell surface localization or host mucin oligosaccharide adhesion mechanism. Our study constitutes the first multiscale computational study to explore the plasminogen receptor function of GiENO for its further consideration as a potential therapeutic target for giardiasis treatment.

Stem cells offer great promise within the field of regenerative medicine but despite encouraging results, the large scale use of stem cells for therapeutic applications still faces challenges when it comes to controlling signaling pathway responses with respect to environmental perturbations. This step is critical for the elaboration of stable and reproducible differentiation protocols, and computational modeling can be helpful to overcome these difficulties. This article is a comparative review of the mechanism-based methods used for hypothesis-driven approaches and data-driven methods which are two types of computational approaches commonly used for analysing the dynamics of pathways involved in stem cell regulation. We firstly review works based on kinetic modelling. We emphasize the relationships between the dynamics of these pathways and their topological features, and illustrative examples are described to show how the analysis of these relationships can contribute to a more detailed and formal understanding of the signaling dynamics. This discussion is followed by a review of the recent data-driven pathway analysis methods. Based on a simplified description of the pathways, these methods are able to handle high dimensionality data, and topological features of the pathways taken into account in the latest methods improve both accuracy and predictive power. Nevertheless, progress is still needed to clarify the biological meaning of the topological decompositions used by these methods.

The toxic action of different endogenous and exogenous agents leads to damage in genomic DNA. 8-Oxoguanine is one of the most often generated and highly mutagenic oxidative forms of damage in DNA. Normally, in human cells it is promptly removed by 8-oxoguanine-DNA-glycosylase hOGG1, the key DNA-repair enzyme. An association between the accumulation of oxidized guanine and an increased risk of harmful processes in organisms was already found. However, the detailed mechanism of damaged base recognition and removal is still unclear. To clarify the role of active site amino acids in the damaged base coordination and to reveal the elementary steps in the overall enzymatic process we investigated hOGG1 mutant forms with substituted amino acid residues in the enzyme base-binding pocket. Replacing the functional groups of the enzyme active site allowed us to change the rates of the individual steps of the enzymatic reaction. To gain further insight into the mechanism of hOGG1 catalysis a detailed pre-steady state kinetic study of this enzymatic process was carried out using the stopped-flow approach. The changes in the DNA structure after mixing with enzymes were followed by recording the FRET signal using Cy3/Cy5 labels in DNA substrates in the time range from milliseconds to hundreds of seconds. DNA duplexes containing non-damaged DNA, 8-oxoG, or an AP-site or its unreactive synthetic analogue were used as DNA-substrates. The kinetic parameters of DNA binding and damage processing were obtained for the mutant forms and for WT hOGG1. The analyses of fluorescence traces provided information about the DNA dynamics during damage recognition and removal. The kinetic study for the mutant forms revealed that all introduced substitutions reduced the efficiency of the hOGG1 activity; however, they played pivotal roles at certain elementary stages identified during the study. Taken together, our results gave the opportunity to restore the role of substituted amino acids and main “damaged base–amino acid” contacts, which provide an important link in the understanding the mechanism of the DNA repair process catalyzed by hOGG1.

Non-Alcoholic Fatty Liver Disease (NAFLD) is a complex spectrum of diseases ranging from simple steatosis to Non-Alcoholic Steatohepatitis (NASH) with fibrosis, which can progress to cirrhosis and hepatocellular carcinoma. The pathogenesis of NAFLD is complex, involving crosstalk between multiple organs, cell-types, and environmental and genetic factors. Dysfunction of the adipose tissue plays a central role in NAFLD progression. Here, we analysed transcriptomics data obtained from the Visceral Adipose Tissue (VAT) of NAFLD patients to understand how the VAT metabolism is altered at the genome scale and co-regulated with other cellular processes during the progression from obesity to NASH with fibrosis. For this purpose, we performed Weighted Gene Co-expression Network Analysis (WGCNA), a method that organizes the disease transcriptome into functional modules of cellular processes and pathways. Our analysis revealed the coordination of metabolic and inflammatory modules (termed “immunometabolism”) in the VAT of NAFLD patients. We found that genes of arachidonic acid, sphingolipid and glycosphingolipid metabolism were upregulated and co-expressed with genes of proinflammatory signalling pathways and hypoxia in NASH/NASH with fibrosis. We hypothesize that these metabolic alterations might play a role in sustaining VAT inflammation. Furthermore, immunometabolism related genes were also co-expressed with genes involved in Extracellular Matrix (ECM) degradation. Our analysis indicates that upregulation of both ECM degrading enzymes and their inhibitors (incoherent feedforward loop) potentially leads to the ECM deposition in the VAT of NASH with fibrosis patients.