Protein technologies and commercial enzymes

A selection of World Wide Web sites relevant to papers published in this issue of Current Opinion in Biotechnology.     

Protein technologies and commercial enzymes

A selection of World Wide Web sites relevant to papers published in this issue of Current Opinion in Biotechnology.     

Protein interactomics based on direct molecular fishing on paramagnetic particles: Experimental simulation and SPR validation

We describe an experimental approach for direct molecular fishing of prey protein on the surface of two types of paramagnetic particles (PMP) having different size and composition. Human microsomal cytochrome b₅ (b₅) and its known partner human cytochrome P450 3A5 (CYP3A5) were used as bait and prey proteins, respectively. For assessing the level of unspecific binding of background proteins, α‐fetoprotein (aFP) was used. SPR measurements were applied for quantitative analysis of trapped proteins (CYP3A5 and aFP) after fishing on PMP. It was shown that the described approach of molecular fishing on micro‐PMP provides enough prey proteins for LC‐MS/MS identification and SPR validation, so this approach can be used for discovery of new protein–protein interactions in the framework of Human Proteome Project.     

Protein interactomics based on direct molecular fishing on paramagnetic particles: Practical realization and further SPR validation

There is increasing evidence that proteins function in the cell as integrated stable or temporally formed protein complexes, interactomes. Previously, using model systems we demonstrated applicability of direct molecular fishing on paramagnetic particles for protein interactomics (Ershov et al. Proteomics, 2012, 12, 3295). In the present study, we have used a combination of affinity‐based molecular fishing and subsequent MS for investigation of human liver proteins involved in interactions with immobilized microsomal cytochrome b5 (CYB5A), and also transthyretin and BSA as alternative affinity ligands (baits). The LC?MS/MS identification of prey proteins fished on these baits revealed three sets of proteins: 98, 120, and 220, respectively. Comparison analysis of these sets revealed only three proteins common for all the baits. In the case of paired analysis, the number of common proteins varied from 2 to 9. The binding capacity of some identified proteins has been validated by a SPR‐based biosensor. All the investigated proteins effectively interacted with the immobilized CYB5A (Kd values ranged from 0.07 to 1.1 μM). Results of this study suggest that direct molecular fishing is applicable for analysis of protein–protein interactions (PPI) under normal and pathological conditions, in which altered PPIs are especially important.     

Comparative protein interactomics of neuroglobin and myoglobin

Neuroglobin is a hypoxia-inducible O(2) -binding protein with neuroprotective effects in cell and animal models of stroke and Alzheimer's disease. The mechanism underlying neuroglobin's cytoprotective action is unknown, although several possibilities have been proposed, including anti-oxidative and anti-apoptotic effects. We used affinity purification-mass spectrometry methods to identify neuroglobin-interacting proteins in normoxic and hypoxic murine neuronal (HN33) cell lysates, and to compare these interactions with those of a structurally and functionally related protein, myoglobin. We report that the protein interactomes of neuroglobin and myoglobin overlap substantially and are modified by hypoxia. In addition, neuroglobin-interacting proteins include partners consistent with both anti-oxidative and anti-apoptotic functions, as well as with a relationship to several neurodegenerative diseases.     

Reverse interactomics: decoding protein-protein interactions with combinatorial peptide libraries

Identification of binding partners is the crucial first step towards understanding the biological function of a protein. Many protein-protein interactions occur via modular domains that recognize short peptide motifs in their target proteins. Here we describe a chemical/bioinformatics approach for predicting the binding partners of modular domains. The optimal binding motif(s) of a protein domain is identified by screening a combinatorial peptide library. The resulting consensus sequence is used to search protein and genomic databases for potential binding proteins, which are subsequently confirmed (or disproved) by conventional protein binding assays (e.g. pull-down and co-immunoprecipitation).     

Protein and lysate array technologies in cancer research

Capturing quantitative proteomic information provides new insights for enhancing the understanding of cancer biology. There have been several protein microarray formats, and each has an advantage depending on what is being detected. However, in contrast to nucleotide printing, the production of protein arrays generally requires the capability of handling viscous solutions, and the mishandling of various factors, such as temperature and humidity, adversely affect protein status. The requirement for such specifications is critical when increasing the throughput for monitoring a large number of samples for rigorous quantitation. In particular, a new solid pin arrayer has been extremely powerful when highly viscous cell lysates printed for high-density, "reverse-phase" protein arrays, and acquired data allows for theoretical models of protein signaling networks to be constructed. In this review, applications of currently available protein microarray technology to cancer research are discussed including the advantages of the new solid pin architecture for opening up powerful proteomic applications.     

Pathways involved in viral oncogenesis: New perspectives from virus-host protein interactomics

Oncogenic viruses are among the apparent causes of cancer-associated mortality. It was estimated that 12% to 15% of human malignancies are linked to oncoviruses. Although modernist strategies and traditional genetic studies have defined host-pathogen interactions of the oncoviruses, their host functions which are critical for the establishment of infection still remain mysterious. However, over the last few years, it has become clear that infections hijack and modify cellular pathways for their benefit. In this context, we constructed the virus-host protein interaction networks of seven oncoviruses (EBV, HBV, HCV, HTLV-1, HHV8, HPV16, and HPV18), and revealed cellular pathways hijacking as a result of oncogenic virus infection. Several signaling pathways/processes such as TGF-β signaling, cell cycle, retinoblastoma tumor suppressor protein, and androgen receptor signaling were mutually targeted by viruses to induce oncogenesis. Besides, cellular pathways specific to a certain virus were detected. By this study, we believe that we improve the understanding of the molecular pathogenesis of viral oncogenesis and provide information in setting new targets for treatment, prognosis, and diagnosis.     

Protein fibers as performance proteins: new technologies and applications

Protein fibers are fundamental building blocks of life playing an essential role in motility, elasticity, scaffolding, stabilization and the protection of cells, tissues and organisms. Despite nearly a century of research into the assembly mechanisms and structures of fibrous proteins, only limited information is still available. Within the past decade, however, insights have been provided into how some fibrous proteins assemble and how they function in biology. In addition, efforts are increasingly being made to employ protein fibers as performance molecules in man-made medical and technical applications.     

Native mass spectrometry: a bridge between interactomics and structural biology

Native mass spectrometry is an emerging technology that allows the topological investigation of intact protein complexes with high sensitivity and a theoretically unrestricted mass range. This unique tool provides complementary information to established technologies in structural biology, and also provides a link to high-throughput interactomics studies, which do not generate information on exact protein complex-composition, structure or dynamics. Here I review the current state of native mass spectrometry technology and discuss several important biological applications. I also describe current experimental challenges in native mass spectrometry, encouraging readers to contribute to solutions.