Molecular glues to stabilise protein–protein interactions

Targeting protein–protein interactions (PPIs) has become a common approach to tackle various diseases whose pathobiology is driven by their mis-regulation in important signalling pathways. Modulating PPIs has tremendous untapped therapeutic potential and different approaches can be used to modulate PPIs. Initially, therapeutic effects were mostly sought by inhibiting PPIs. However, by gaining insight in the mode of action of certain therapeutic compounds, it became clear that stabilising (i.e. enhancing) PPIs can also be useful. The latter strategy is recently gaining a lot of attention, as stabilising physiologic, or even inducing novel interactions of a target protein with E3 ubiquitin ligases forms the basis of the targeted protein degradation (TPD) approach. An emerging additional example for drug discovery based on PPI stabilisation are the 14-3-3 proteins, a family of regulatory proteins, which engages in many protein–protein interactions, some of which might become therapeutical targets.     

Druggable protein–protein interactions – from hot spots to hot segments

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From protein structure to biochemical function?

Here we describe various methods currently under development aimed at identifying a proteins function from its three-dimensional structure. We are combining a number of these methods to create a pipeline of applications, called ProFunc, which will take a given 3D structure, run all the applications on it and compile and summarise the results obtained. The aim is to provide a best guess as to the proteins function from the evidence provided by the different methods. Here we present three examples, using structures solved by the Midwest Center for Structural Genomics consortium, illustrating the strengths and weaknesses of current approaches.     

Mass spectrometry-based approaches to protein–ligand interactions

One of the greatest current challenges in proteomics is to develop an understanding of cellular communication and regulation processes, most of which involve noncovalent interactions of proteins with various binding partners. Mass spectrometry plays an important role in all aspects of these research efforts. This article provides a survey of mass spectrometry-based approaches for exploring protein–ligand interactions. A wide array of techniques is available, and the choice of method depends on the specific problem at hand. For example, the high-throughput screening of compound libraries for binding to a specific receptor requires different approaches than structural studies on multiprotein complexes. This review is directed to readers wishing to obtain a concise yet comprehensive overview of existing experimental techniques. Specific emphasis is placed on emerging methods that have been developed within the last few years.     

Mapping the protein–protein and genetic interactions of cancer to guide precision medicine

Massive efforts to sequence cancer genomes have compiled an impressive catalogue of cancer mutations, revealing the recurrent exploitation of a handful of ‘hallmark cancer pathways’. However, unraveling how sets of mutated proteins in these and other pathways hijack pro-proliferative signaling networks and dictate therapeutic responsiveness remains challenging. Here, we show that cancer driver protein–protein interactions are enriched for additional cancer drivers, highlighting the power of physical interaction maps to explain known, as well as uncover new, disease-promoting pathway interrelationships. We hypothesize that by systematically mapping the protein–protein and genetic interactions in cancer—thereby creating Cancer Cell Maps—we will create resources against which to contextualize a patient’s mutations into perturbed pathways/complexes and thereby specify a matching targeted therapeutic cocktail.     

Integrin β4 regulates SPARC protein to promote invasion

The α6β4 integrin (referred to as "β4" integrin) is a receptor for laminins that promotes carcinoma invasion through its ability to regulate key signaling pathways and cytoskeletal dynamics. An analysis of published Affymetrix GeneChip data to detect downstream effectors involved in β4-mediated invasion of breast carcinoma cells identified SPARC, or secreted protein acidic and rich in cysteine. This glycoprotein has been shown to play an important role in matrix remodeling and invasion. Our analysis revealed that manipulation of β4 integrin expression and signaling impacted SPARC expression and that SPARC facilitates β4-mediated invasion. Expression of β4 in β4-deficient cells reduced the expression of a specific microRNA (miR-29a) that targets SPARC and impedes invasion. In cells that express endogenous β4, miR-29a expression is low and β4 ligation facilitates the translation of SPARC through a TOR-dependent mechanism. The results obtained in this study demonstrate that β4 can regulate SPARC expression and that SPARC is an effector of β4-mediated invasion. They also highlight a potential role for specific miRNAs in executing the functions of integrins.     

Can copper binding to the prion protein generate a misfolded form of the protein?

The native prion protein (PrP) has a two domain structure, with a globular folded α-helical C-terminal domain and a flexible extended N-terminal region. The latter can selectively bind Cu²⁺ via four His residues in the octarepeat (OR) region, as well as two sites (His96 and His111) outside this region. In the disease state, the folded C-terminal domain of PrP undergoes a conformational change, forming amorphous aggregates high in β-sheet content. Cu²⁺ bound to the ORs can be redox active and has been shown to induce cleavage within the OR region, a process requiring conserved Trp residues. Using computational modeling, we have observed that electron transfer from Trp residues to copper can be favorable. These models also reveal that an indole-based radical cation or Cu⁺ can initiate reactions leading to protein backbone cleavage. We have also demonstrated, by molecular dynamics simulations, that Cu²⁺ binding to the His96 and His111 residues in the remaining PrP N-terminal fragment can induce localized β-sheet structure, allowing us to suggest a potential mechanism for the initiation of β-sheet misfolding in the C-terminal domain by Cu²⁺.

LEVELNET to visualize,explore, and compare protein–protein interaction networks

Physical interactions between proteins are central to all biological processes. Yet, the current knowledge of who interacts with whom in the cell and in what manner relies on partial, noisy, and highly heterogeneous data. Thus, there is a need for methods comprehensively describing and organizing such data. LEVELNET is a versatile and interactive tool for visualizing, exploring, and comparing protein–protein interaction (PPI) networks inferred from different types of evidence. LEVELNET helps to break down the complexity of PPI networks by representing them as multi-layered graphs and by facilitating the direct comparison of their subnetworks toward biological interpretation. It focuses primarily on the protein chains whose 3D structures are available in the Protein Data Bank. We showcase some potential applications, such as investigating the structural evidence supporting PPIs associated to specific biological processes, assessing the co-localization of interaction partners, comparing the PPI networks obtained through computational experiments versus homology transfer, and creating PPI benchmarks with desired properties.     

Unlocking the secrets to protein–protein interface drug targets using structural mass spectrometry techniques

Protein–protein interactions (PPIs) drive all biologic systems at the subcellular and extracellular level. Changes in the specificity and affinity of these interactions can lead to cellular malfunctions and disease. Consequently, the binding interfaces between interacting protein partners are important drug targets for the next generation of therapies that block such interactions. Unfortunately, protein–protein contact points have proven to be very difficult pharmacological targets because they are hidden within complex 3D interfaces. For the vast majority of characterized binary PPIs, the specific amino acid sequence of their close contact regions remains unknown. There has been an important need for an experimental technology that can rapidly reveal the functionally important contact points of native protein complexes in solution. In this review, experimental techniques employing mass spectrometry to explore protein interaction binding sites are discussed. Hydrogen–deuterium exchange, hydroxyl radical footprinting, crosslinking and the newest technology protein painting are compared and contrasted.     

High throughput platform to explore RNA–protein interactomes

RNA binding proteins (RBPs) and RNA interaction is an emerging topic in molecular biology. Many reports showed that such interactions contribute to many cellular processes as well as disease development. Several standard in vitro and in vivo methods were developed to fulfill the needs of this RBP–RNA interaction study to explore their biological functions. However, these methods have their limitations in terms of throughput. In this review, we emphasize two important high throughput methods to studying RBP–RNA interactions, affinity purification and protein microarray. These methods have recently become robust techniques regarding their efficiency in systematically analyzing RBP–RNA interactions. Here, we provide technique overviews, strategies and applications of these methods during biological research. Although these technologies are just beginning to be explored, they will be most important methods in this study.     

Did evolution leap to create the protein universe?

The genomes of over 60 organisms from all three kingdoms of life are now entirely sequenced. In many respects, the inventory of proteins used in different kingdoms appears surprisingly similar. However, eukaryotes differ from other kingdoms in that they use many long proteins, and have more proteins with coiled-coil helices and with regions abundant in regular secondary structure. Particular structural domains are used in many pathways. Nevertheless, one domain tends to occur only once in one particular pathway. Many proteins do not have close homologues in different species (orphans) and there could even be folds that are specific to one species. This view implies that protein fold space is discrete. An alternative model suggests that structure space is continuous and that modern proteins evolved by aggregating fragments of ancient proteins. Either way, after having harvested proteomes by applying standard tools, the challenge now seems to be to develop better methods for comparative proteomics.     

Why the move to microfluidics for protein analysis?

There has been a recent trend towards the miniaturization of analytical tools, but what are the advantages of microfluidic devices and when is their use appropriate? Recent advances in the field of micro-analytical systems can be classified according to instrument performance (which refers here to the desired property of the analytical tool of interest) and two important features specifically related to miniaturisation, namely reduction of the sample volume and the time-to-result. Here we discuss the contribution of these different parameters and aim to highlight the factors of choice in the development and use of microfluidic devices dedicated to protein analysis.     

Apparent dependence of protein evolutionary rate on number of interactions is linked to biases in protein–protein interactions data sets

Background  

Several studies have suggested that proteins that interact with more partners evolve more slowly. The strength and validity of this association has been called into question. Here we investigate how biases in high-throughput protein–protein interaction studies could lead to a spurious correlation.     

A plasmid-based expression system to study protein–protein interactions at the Golgi in vivo

There is still an unmet need for simple methods to verify, visualize, and confirm protein–protein interactions in vivo. Here we describe a plasmid-based system to study such interactions. The system is based on the transmembrane domain (TMD) of the EF-hand Ca²⁺ sensor protein calneuron-2. We show that fusion of 28 amino acids that include the TMD of calneuron-2 to proteins of interest results in prominent localization on the cytoplasmic side of the Golgi. The recruitment of binding partners to the protein of interest fused to this sequence can then be easily visualized by fluorescent tags.     

In planta analysis of protein–protein interactions related to light signaling by bimolecular fluorescence complementation

The determination of protein-protein interactions is becoming more and more important in the molecular analysis of signal transduction chains. To this purpose the application of a manageable and simple assay in an appropriate biological system is of major concern. Bimolecular fluorescence complementation (BiFC) is a novel method to analyze protein-protein interactions in vivo. The assay is based on the observation that N- and C-terminal subfragments of the yellow-fluorescent protein (YFP) can only reconstitute a functional fluorophore when they are brought into tight contact. Thus, proteins can be fused to the YFP subfragments and the interaction of the fusion proteins can be monitored by epifluorescence microscopy. Pairs of interacting proteins were tested after transient cotransfection in etiolated mustard seedlings, which is a well characterized plant model system for light signal transduction. BiFC could be demonstrated with the F-box protein EID1 (empfindlicher im dunkelroten Licht 1) and the Arabidopsis S-phase kinase-related protein 1 (ASK1). The interaction of both proteins was specific and strictly dependent on the presence of an intact F-box domain. Our studies also demonstrate that etiolated mustard seedlings provide a versatile transient assay system to study light-induced subcellular localization events.