The integrated analysis of metabolic and protein interaction networks reveals novel molecular organizing principles

Background: The study of biological interaction networks is a central theme of systems biology. Here, we investigate the relationships between two distinct types of interaction networks: the metabolic pathway map and the protein-protein interaction network (PIN). It has long been established that successive enzymatic steps are often catalyzed by physically interacting proteins forming permanent or transient multi-enzymes complexes. Inspecting high-throughput PIN data, it was shown recently that, indeed, enzymes involved in successive reactions are generally more likely to interact than other protein pairs. In our study, we expanded this line of research to include comparisons of the underlying respective network topologies as well as to investigate whether the spatial organization of enzyme interactions correlates with metabolic efficiency. Results: Analyzing yeast data, we detected long-range correlations between shortest paths between proteins in both network types suggesting a mutual correspondence of both network architectures. We discovered that the organizing principles of physical interactions between metabolic enzymes differ from the general PIN of all proteins. While physical interactions between proteins are generally dissortative, enzyme interactions were observed to be assortative. Thus, enzymes frequently interact with other enzymes of similar rather than different degree. Enzymes carrying high flux loads are more likely to physically interact than enzymes with lower metabolic throughput. In particular, enzymes associated with catabolic pathways as well as enzymes involved in the biosynthesis of complex molecules were found to exhibit high degrees of physical clustering. Single proteins were identified that connect major components of the cellular metabolism and may thus be essential for the structural integrity of several biosynthetic systems. Conclusion: Our results reveal topological equivalences between the protein interaction network and the metabolic pathway network. Evolved protein interactions may contribute significantly towards increasing the efficiency of metabolic processes by permitting higher metabolic fluxes. Thus, our results shed further light on the unifying principles shaping the evolution of both the functional (metabolic) as well as the physical interaction network

Rapid Diffusion of Green Fluorescent Protein in the Mitochondrial Matrix

Abstract. It is thought that the high protein density in the mitochondrial matrix results in severely restricted solute diffusion and metabolite channeling from one enzyme to another without free aqueous-phase diffusion. To test this hypothesis, we measured the diffusion of green fluorescent protein (GFP) expressed in the mitochondrial matrix of fibroblast, liver, skeletal muscle, and epithelial cell lines. Spot photobleaching of GFP with a 100× objective (0.8-μm spot diam) gave half-times for fluorescence recovery of 15–19 ms with >90% of the GFP mobile. As predicted for aqueous-phase diffusion in a confined compartment, fluorescence recovery was slowed or abolished by increased laser spot size or bleach time, and by paraformaldehyde fixation. Quantitative analysis of bleach data using a mathematical model of matrix diffusion gave GFP diffusion coefficients of 2–3 × 10−7 cm2/s, only three to fourfold less than that for GFP diffusion in water. In contrast, little recovery was found for bleaching of GFP in fusion with subunits of the fatty acid β-oxidation multienzyme complex that are normally present in the matrix. Measurement of the rotation of unconjugated GFP by time-resolved anisotropy gave a rotational correlation time of 23.3 ± 1 ns, similar to that of 20 ns for GFP rotation in water. A rapid rotational correlation time of 325 ps was also found for a small fluorescent probe (BCECF, ∼0.5 kD) in the matrix of isolated liver mitochondria. The rapid and unrestricted diffusion of solutes in the mitochondrial matrix suggests that metabolite channeling may not be required to overcome diffusive barriers. We propose that the clustering of matrix enzymes in membrane-associated complexes might serve to establish a relatively uncrowded aqueous space in which solutes can freely diffuse

Development of cross metathesis for the design of HDAC inhibitors

Epigenetic histones post translational modifications are key players in the regulation of gene expression. Amongst all possible modifications, the N-acetylation of the lysine side chains located at the N-terminal tail of histones is involved in the relaxation of chromatine and contributes to gene transcription. This acetylation status is controlled by histone acetyl transferases (HAT) and histone deacetylases (HDAC and SIRT). The abnormal expression of HDAC has been linked to the progression of several human diseases such as cancers. Therefore, the development of HDAC inhibitors has emerged as a valuable therapeutic strategy, with currently four compounds approved by the FDA and a fifth one in China. Recent data suggested that combination therapies with HDAC inhibitors may contribute to better clinical results. HDACs are zinc-dependant enzymes grouped in a family of 11 proteins (HDAC1-11), grouped in three classes: class I (HDAC1-3,8), class Il (HDAC4-7,9,10) and class IV (HDAC11). The fourth group of deacetylases contains the class Ill NADH+-dependent sirtuins (SIRT1-7). The standard pharmacophore for HDAC inhibitors involves a zinc-binding group (ZBG) linked through a spacer to a “cap” group in interaction with the external solvent accessible surface. We were interested in alternative chemistries to access HDAC inhibitors in which the intermediate spacer could be build out of two similar building blocks, one bearing the ZBG and on bearing the cap group. By selecting linear alkyl-based spacer, cross metathesis appeared to be a short and flexible strategy, provided it supports the presence of highly oxophile functional groups such as carbonyl groups. This poster summarizes our findings in the application of cross metathesis and the biological evaluations of some of the compounds obtained

Towards a matrix mechanics framework for dynamic protein network

Protein–protein interaction networks are currently visualized by software generated interaction webs based upon static experimental data. Current state is limited to static, mostly non-compartmental network and non time resolved protein interactions. A satisfactory mathematical foundation for particle interactions within a viscous liquid state (situation within the cytoplasm) does not exist nor do current computer programs enable building dynamic interaction networks for time resolved interactions. Building mathematical foundation for intracellular protein interactions can be achieved in two increments (a) trigger and capture the dynamic molecular changes for a select subset of proteins using several model systems and high throughput time resolved proteomics and, (b) use this information to build the mathematical foundation and computational algorithm for a compartmentalized and dynamic protein interaction network. Such a foundation is expected to provide benefit in at least two spheres: (a) understanding physiology enabling explanation of phenomenon such as incomplete penetrance in genetic disorders and (b) enabling several fold increase in biopharmaceutical production using impure starting materials

Yeast Two-Hybrid, a Powerful Tool for Systems Biology

A key property of complex biological systems is the presence of interaction networks formed by its different components, primarily proteins. These are crucial for all levels of cellular function, including architecture, metabolism and signalling, as well as the availability of cellular energy. Very stable, but also rather transient and dynamic protein-protein interactions generate new system properties at the level of multiprotein complexes, cellular compartments or the entire cell. Thus, interactomics is expected to largely contribute to emerging fields like systems biology or systems bioenergetics. The more recent technological development of high-throughput methods for interactomics research will dramatically increase our knowledge of protein interaction networks. The two most frequently used methods are yeast two-hybrid (Y2H) screening, a well established genetic in vivo approach, and affinity purification of complexes followed by mass spectrometry analysis, an emerging biochemical in vitro technique. So far, a majority of published interactions have been detected using an Y2H screen. However, with the massive application of this method, also some limitations have become apparent. This review provides an overview on available yeast two-hybrid methods, in particular focusing on more recent approaches. These allow detection of protein interactions in their native environment, as e.g. in the cytosol or bound to a membrane, by using cytosolic signalling cascades or split protein constructs. Strengths and weaknesses of these genetic methods are discussed and some guidelines for verification of detected protein-protein interactions are provided

Synthetic biology approaches in drug discovery and pharmaceutical biotechnology

Synthetic biology is the attempt to apply the concepts of engineering to biological systems with the aim to create organisms with new emergent properties. These organisms might have desirable novel biosynthetic capabilities, act as biosensors or help us to understand the intricacies of living systems. This approach has the potential to assist the discovery and production of pharmaceutical compounds at various stages. New sources of bioactive compounds can be created in the form of genetically encoded small molecule libraries. The recombination of individual parts has been employed to design proteins that act as biosensors, which could be used to identify and quantify molecules of interest. New biosynthetic pathways may be designed by stitching together enzymes with desired activities, and genetic code expansion can be used to introduce new functionalities into peptides and proteins to increase their chemical scope and biological stability. This review aims to give an insight into recently developed individual components and modules that might serve as parts in a synthetic biology approach to pharmaceutical biotechnology