Mass spectrometry informs the structure and dynamics of membrane proteins involved in lipid and drug transport

Membrane proteins are important macromolecules that play crucial roles in many cellular and physiological processes. Over the past two decades, the use of mass spectrometry as a biophysical tool to characterise membrane proteins has grown steadily. By capturing these dynamic complexes in the gas phase, many unknown small molecule interactions have been revealed. One particular application of this research has been the focus on antibiotic resistance with considerable efforts being made to understand underlying mechanisms. Here we review recent advances in the application of mass spectrometry that have yielded both structural and dynamic information on the interactions of antibiotics with proteins involved in bacterial cell envelope biogenesis and drug efflux.     

Tools used to study how protein complexes are assembled in signaling cascades

Most proteins do not function on their own but as part of large signaling complexes that are arranged in every living cell in response to specific environmental cues. Proteins interact with each other either constitutively or transiently and do so with different affinity. When identifying the role played by a protein inside a cell, it is essential to define its particular cohort of binding partners so that the researcher can predict what signaling pathways the protein is engaged in. Once identified and confirmed, the information might allow the interaction to be manipulated by pharmacological inhibitors to help fight disease. In this review, we discuss protein-protein interactions and how they are essential to propagate signals in signaling pathways. We examine some of the high-throughput screening methods and focus on the methods used to confirm specific protein-protein interactions including; affinity tagging, co-immunoprecipitation, peptide array technology and fluorescence microscopy.     

PepBind: A Comprehensive Database and Computational Tool for Analysis of Protein-peptide Interactions

Protein–peptide interactions, where one partner is a globular protein (domain) and the other is a flexible linear peptide, are key components of cellular processes predominantly in signaling and regulatory networks, hence are prime targets for drug design. To derive the details of the protein–peptide interaction mechanism is often a cumbersome task, though it can be made easier with the availability of specific databases and tools. The Peptide Binding Protein Database (PepBind) is a curated and searchable repository of the structures, sequences and experimental observations of 3100 protein–peptide complexes. The web interface contains a computational tool, protein inter-chain interaction (PICI), for computing several types of weak or strong interactions at the protein–peptide interaction interface and visualizing the identified interactions between residues in Jmol viewer. This initial database release focuses on providing protein–peptide interface information along with structure and sequence information for protein–peptide complexes deposited in the Protein Data Bank (PDB). Structures in PepBind are classified based on their cellular activity. More than 40% of the structures in the database are found to be involved in different regulatory pathways and nearly 20% in the immune system. These data indicate the importance of protein–peptide complexes in the regulation of cellular processes. PepBind is freely accessible at http://pepbind.bicpu.edu.in/.     

RNA-Mediated Disease Mechanisms in Neurodegenerative Disorders

RNA is accurately entangled in virtually all pathways that maintain cellular homeostasis. To name but a few, RNA is the “messenger” between DNA encoded information and the resulting proteins. Furthermore, RNAs regulate diverse processes by forming DNA::RNA or RNA::RNA interactions. Finally, RNA itself can be the scaffold for ribonucleoprotein complexes, for example, ribosomes or cellular bodies. Consequently, disruption of any of these processes can lead to disease. This review describes known and emerging RNA-based disease mechanisms like interference with regular splicing, the anomalous appearance of RNA–protein complexes and uncommon RNA species, as well as non-canonical translation. Due to the complexity and entanglement of the above-mentioned pathways, only few drugs are available that target RNA-based disease mechanisms. However, advances in our understanding how RNA is involved in and modulates cellular homeostasis might pave the way to novel treatments.     

Scaffold Proteins IRSp53 and Spinophilin Regulate Localized Rac Activation by T-lymphocyte Invasion and Metastasis Protein 1 (TIAM1)

The Rac exchange factor Tiam1 is involved in diverse cell functions and signaling pathways through multiple protein interactions, raising the question of how signaling and functional specificity are achieved. We have shown that Tiam1 interactions with different scaffold proteins activate different Rac-dependent pathways by recruiting specific Rac effector proteins, and reasoned that there must be regulatory mechanisms governing each interaction. Fibroblasts express at least two Tiam1-interacting proteins, insulin receptor substrate protein 53 kDa (IRSp53) and spinophilin. We used fluorescent resonance energy transfer (FRET) to measure localized Rac activation associated with IRSp53 and spinophilin complexes in individual fibroblasts to test this hypothesis. Pervanadate or platelet-derived growth factor induced localized Rac activation dependent on Tiam1 and IRSp53. Forskolin or epinephrine induced localized Rac activation dependent on Tiam1 and spinophilin. In spinophilin-deficient cells, Tiam1 co-localized with IRSp53 in response to pervanadate or platelet-derived growth factor. In IRSp53-deficient cells, Tiam1 co-localized with spinophilin in response to forskolin or epinephrine. Total cellular levels of activated Rac were affected only in cells with exogenous Tiam1, and were primarily increased in the membrane fraction. Downstream effects of Rac activation were also stimulus and scaffold-specific. Cell ruffling, spreading, and cell adhesion were dependent on IRSp53, but not spinophilin. Epinephrine decreased IRSp53-dependent adhesion and increased cell migration in a Rac and spinophilin-dependent fashion. These results support the idea that Tiam1 interactions with different scaffold proteins couple distinct upstream signals to localized Rac activation and specific downstream pathways, and suggest that manipulating Tiam1-scaffold interactions can modulate Rac-dependent cellular behaviors.     

Protein interaction data set highlighted with human Ras-MAPK/PI3K signaling pathways

The Ras-MAPK and PI3K-AKT pathways are conserved in metazoan organisms, which involve a series of signaling cascades and form the basis for numerous physiological and pathological processes. Here we report on yeast two hybrid screening results of a protein interaction network around the known components of human Ras-MAPK/PI3K pathways. A total of 42 independent cDNA library screenings resulted in 200 protein-protein interaction (PPI) pairs among 180 molecules. Most of the proteins formed a large cluster that contains 193 PPIs between 169 proteins. Seventy-four interactions indicate high-confidence according to bioinformatics analysis. The prey list contains high enrichment genes with specific Gene Ontology (GO) terms such as response to stress and response to external stimulus. Most interactions link the Ras signaling pathway with various cellular processes. Five interactions were validated by coimmunoprecipitation and colocalization assays in mammalian cells to confirm their in vivo interactions. This protein interaction network provides further insights into the molecular mechanism of Ras-MAPK/PI3K signaling pathways.     

Protein myristoylation in protein-lipid and protein-protein interactions

Various proteins in signal transduction pathways are myristoylated. Although this modification is often essential for the proper functioning of the modified protein, the mechanism by which the modification exerts its effects is still largely unknown. Here we discuss the roles played by protein myristoylation, in both protein-lipid and protein-protein interactions. Myristoylation is involved in the membrane interactions of various proteins, such as MARCKS and endothelial NO synthase. The intermediate hydrophobic nature of the modification plays an important role in the reversible membrane anchoring of these proteins. The anchoring is strengthened by a basic amphiphilic domain that works as a switch for the reversible binding. Protein myristoylation is also involved in protein-protein interactions, which are regulated by the interplay between protein phosphorylation, calmodulin binding, and membrane phospholipids.     

Targeting secret handshakes of biological processes for novel drug development

In multicellular organisms, several biological processes control the rise and fall of life. Different cell types communicate and co-operate in response to different stimulus through cell to cell signaling and regulate biologic processes in the cell/organism. Signaling in multicellular organism has to be made very secretly so that only the target cell responds to the signal. Of all the biomolecules, nature chose mainly proteins for secret delivery of information both inside and outside the cell. During cell signaling, proteins physically interact and shake hands for transfer of secret information by a phenomenon called as protein–protein interactions (PPIs). In both, extra and intracellular signaling processes PPIs play a crucial role. PPIs involved in cellular signaling are the primary cause for cell proliferation, differentiation, movement, metabolism, death and various other biological processes not mentioned here. These secret handshakes are very specific for specific functions. Any alterations/malfunctions in particular PPIs results in diseased condition. An overview of signaling pathways and importance of PPIs in cellular function and possibilities of targeting PPIs for novel drug development are discussed in this review.     

The molecular basis of shoot responses of maize seedlings to Trichoderma harzianum T22 inoculation of the root: a proteomic approach

Trichoderma spp. are effective biocontrol agents for several soil-borne plant pathogens, and some are also known for their abilities to enhance systemic resistance to plant diseases and overall plant growth. Root colonization with Trichoderma harzianum Rifai strain 22 (T22) induces large changes in the proteome of shoots of maize (Zea mays) seedlings, even though T22 is present only on roots. We chose a proteomic approach to analyze those changes and identify pathways and genes that are involved in these processes. We used two-dimensional gel electrophoresis to identify proteins that are differentially expressed in response to colonization of maize plants with T22. Up- or down-regulated spots were subjected to tryptic digestion followed by identification using matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry and nanospray ion-trap tandem mass spectrometry. We identified 91 out of 114 up-regulated and 30 out of 50 down-regulated proteins in the shoots. Classification of these revealed that a large portion of the up-regulated proteins are involved in carbohydrate metabolism and some were photosynthesis or stress related. Increased photosynthesis should have resulted in increased starch accumulation in seedlings and did indeed occur. In addition, numerous proteins induced in response to Trichoderma were those involved in stress and defense responses. Other processes that were up-regulated were amino acid metabolism, cell wall metabolism, and genetic information processing. Conversely, while the proteins involved in the pathways noted above were generally up-regulated, proteins involved in other processes such as secondary metabolism and protein biosynthesis were generally not affected. Up-regulation of carbohydrate metabolism and resistance responses may correspond to the enhanced growth response and induced resistance, respectively, conferred by the Trichoderma inoculation.     

Abundance and functional roles of intrinsic disorder in the antimicrobial peptides of the NK-lysin family

NK-lysins are antimicrobial peptides (AMPs) that participate in the innate immune response and also have several pivotal roles in various biological processes. Such multifunctionality is commonly found among intrinsically disordered proteins. However, NK-lysins have never been systematically analyzed for intrinsic disorder. To fill this gap, the amino acid sequences of NK-lysins from various species were collected from UniProt and used for the comprehensive computational analysis to evaluate the propensity of these proteins for intrinsic disorder and to investigate the potential roles of disordered regions in NK-lysin functions. We analyzed abundance and peculiarities of intrinsic disorder distribution in all-known NK-lysins and showed that many NK-lysins are expected to have substantial levels of intrinsic disorder. Curiously, high level of intrinsic disorder was also found even in two proteins with known 3D-strucutres (NK-lysin from pig and human granulysin). Many of the identified disordered regions can be involved in protein–protein interactions. In fact, NK-lysins are shown to contain three to eight molecular recognition features; i.e. short structure-prone segments which are located within the long disordered regions and have a potential to undergo a disorder-to-order transition upon binding to a partner. Furthermore, these disordered regions are expected to have several sites of various posttranslational modifications. Our study shows that NK-lysins, which are AMPs with a set of prominent roles in the innate immune response, are expected to abundantly possess intrinsically disordered regions that might be related to multifunctionality of these proteins in the signal transduction pathways controlling the host response to pathogenic agents.     

Dynamic remodeling of Escherichia coli interactome in response to environmental perturbations

Proteins play an essential role in the vital biological processes governing cellular functions. Most proteins function as members of macromolecular machines, with the network of interacting proteins revealing the molecular mechanisms driving the formation of these complexes. Profiling the physiology-driven remodeling of these interactions within different contexts constitutes a crucial component to achieving a comprehensive systems-level understanding of interactome dynamics. Here, we apply co-fractionation mass spectrometry and computational modeling to quantify and profile the interactions of ∼2000 proteins in the bacterium Escherichia coli cultured under 10 distinct culture conditions. The resulting quantitative co-elution patterns revealed large-scale condition-dependent interaction remodeling among protein complexes involved in diverse biochemical pathways in response to the unique environmental challenges. The network-level analysis highlighted interactome-wide biophysical properties and structural patterns governing interaction remodeling. Our results provide evidence of the local and global plasticity of the E. coli interactome along with a rigorous generalizable framework to define protein interaction specificity. We provide an accompanying interactive web application to facilitate the exploration of these rewired networks.     

NMR studies of protein interactions

Interactions of proteins with other macromolecules or small molecules play important roles in most biological processes. Often, such interactions are weak and transient, and the complexes do not easily crystallize. NMR spectroscopy has the unique ability to retrieve information about these interactions and is increasingly used. Recent methodological developments have helped characterize weak protein interactions, and have in particular been applied to the study of proteins that are mostly unfolded alone but form well-defined complexes upon interaction. In addition, NMR methods have been applied to the identification and characterization of small chemicals that inhibit protein function, a primary objective of rational drug design.     

PYRIN domains and their interactions in the apoptosis and inflammation signaling pathway

The PYRIN domain (PYD) is a well known protein interaction module and a prime mediator of the protein interactions necessary for apoptosis, inflammation and innate immune signaling pathway. Because PYD-mediated apoptosis, inflammation and innate immune processes are associated with many human diseases, studies in these areas are of great biological importance. Intensive biochemical and structural studies of PYD have been conducted in the past decade to elucidate PYD-mediated signaling events, and evaluations of the molecular structure of PYDs have shown the underlying molecular basis for the assembly of PYD-mediated complexes and for the regulation of inflammation and innate immunity. This review summarizes the structure and function of various PYDs and proposes a PYD:PYD interaction for assembly of the complexes involved in those signaling pathways.