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Protein technologies
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Protein technologies Editorial overview John F Timms Current Opinion in Biotechnology 2008, 19:313–315 Available online 11th August 2008 0958-1669/$ – see front matter # 2008 Elsevier Ltd. All rights reserved. DOI 10.1016/j.copbio.2008.07.003
John F Timms Cancer Proteomics Laboratory, EGA Institute for Women’s Health, Cruciform Building, University College London, London, UK e-mail: [email protected]
John F Timms, DPhil, is a senior scientist at the EGA Institute for Women’s Health, University College London where he is head of the Cancer Proteomics Laboratory. He has worked at Harvard Medical School and the Ludwig Institute for Cancer Research before taking up his current post at University College London. His research activities are focused on understanding the molecular mechanisms of growth factor and stress-activated signalling in normal and cancer cells and the discovery of cancer biomarkers using proteomics-based approaches and other techniques.
Proteins are the functional representations of the genes which encode them and they regulate and perform almost all biological processes in cells and tissues. Since nearly all coding genes have now been identified using genomic methods, our next challenge is to define the molecular and biological functions of these gene products. This is a daunting task given the unique nature of every protein and all the possible modifications that they are subject to. For this reason, novel post-genomic technologies are required to interrogate protein function on a large scale. An integral part of the functional activity of proteins revolves around their specific interactions with one another. Identifying protein–protein interactions, particularly in the context of different cellular processes or responses, is therefore critical to understanding protein function and hence cell biology. Protein–protein interactions also play a major role in disease and there is great promise in the development of therapeutic agents which can specifically target protein– protein interactions. This section reviews the different technologies that are used for studying protein interactions both in vitro and in vivo and which can be applied on a large, even genome-wide, scale. The reviews include recent methodological advances which are allowing researchers to now define weak or transient protein–protein interactions and the interactions of membrane proteins which have previously been difficult to study due to their nature. Applications of these methodologies in specific areas of cell biology are highlighted, including the study of cellular signalling events and the identification of dynamic changes in protein complexes using quantitative methods. The reviews cover reporter-based systems such as the yeast twohybrid (YTH) technology used for large-scale screening of protein interactions, through to more targeted epitope-tagging, purification and mass spectrometry (MS)-based approaches for characterising the components of multi-protein complexes. The principles and applications of resonance energy transfer (RET) methods for studying specific interactions in vivo is also covered. The last review discusses our current knowledge of structural determinants of protein–protein interactions and tools used for predicting protein interactions in silico. The latter also includes reference to useful resources of protein interaction data and software for interrogating these databases. Finally, the reviews provide a valuable resource for further reading and cover the seminal papers that have contributed to each area of this emerging and exciting field. An important starting point for understanding any protein’s function is knowledge of all possible interaction partners for that protein. In this respect, genome-wide YTH technology can be applied relatively easily with the availability of genomic or cDNA prey libraries expressing all
www.sciencedirect.com
Current Opinion in Biotechnology 2008, 19:313–315
314 Systems biology
encoded or expressed proteins in the organism or tissue of interest. In their review, Suter, Kittanakom and Stagljar provide a thorough account of the basic YTH technology and its many variants. These variants now allow interaction screening in a context that better reflects the physiological setting where the interactions occur. For example, adaptations for studying membrane protein interactions and use in mammalian cell systems are discussed. The review also provides a background on the pitfalls of the technology and how it can be adapted to decipher signalling pathways, to study the cellular compartmentalisation of interactions, to characterise interfaces within protein complexes and for the screening of small molecules with potential therapeutic use. A major drawback of the basic YTH technology is the relatively high number of false negatives and positives found in screens. These arise due to a failure of nuclear localization, improper folding of fusion proteins, a lack of cofactors necessary for interactions, forced overexpression of heterologous proteins and self-activation of the reporter gene by the bait. To partly overcome this problem, Collins and Choudhary outline how various affinity purification strategies can be combined with tandem MS for the identification of protein–protein interactions in more appropriate cellular models. In particular, it describes the design and use of single epitope and tandem affinity purification (TAP) tags, thereby bypassing the difficulties of generating antibodies against each target protein of interest. It discusses the optimisation of purification conditions, experimental design and the data analysis techniques which have been used to improve the sensitivity and specificity of detecting bona fide protein–protein interactions in different model systems. The authors describe seminal work where through the advent of high accuracy and high throughput MS instrumentation it is now possible to perform near proteome-wide analyses of affinity-purified multi-protein complexes, and when combined with MS-based quantification methods such as SILAC, it has become possible to study the dynamics of protein–protein interactions on a large scale. The authors also discuss recently developed methods for the identification of transient protein–protein interactions and introduce the subject of discriminating specific from non-specific binders. The problem of specificity and the detection of weak or transient interactions using affinity purification and MSbased approaches are further covered in the review by Vermeulen, Hubner and Mann. Whilst extensive purification is required to identify specific interactions, this comes at the cost of reduced throughput and the potential failure in detecting the weak interactors in protein complexes. After reviewing state-of-the-art MS-based quantification methods, the authors describe recently developed stable isotope-based quantitative proteomic methods which are being used to directly compare specific and Current Opinion in Biotechnology 2008, 19:313–315
control (single-step) pull downs. Specific interactors are revealed by their quantitative ratios (heavy versus light tag) and can be retrieved from the excess of non-specific background proteins. However, the authors warn that exchange of dynamic interaction partners can occur during incubation of mixed heavy and light-labelled samples and therefore must be controlled. They also describe recent work where mass tagging has been combined with RNAi for the generation of control samples to study endogenous protein complexes by immunoprecipitation, rather than using overexpressed epitope-tagged proteins and non-immune or ‘bead-only’ controls. Studies using isotopically labelled cross-linkers are also described where the aim is to stabilise and quantify multi-protein complexes. The application of these methods has proven successful in the identification of novel and post-translational modification-specific protein complexes in tyrosine kinase signalling, cell adhesion and chromatin biology. The review by Ciruela next focuses on non-invasive fluorescence-based methods using resonance energy transfer (RET). These technologies, namely Bioluminescence-RET(BRET) and Fluorescence-RET (FRET), and those centred on protein fragment complementation, such as Bimolecular Fluorescence Complementation (BiFC), have been successfully applied for visualising interactions occurring within protein complexes in living cells. The individual strengths and weaknesses of these methods when applied to the study of protein–protein interactions are discussed. Although these methods are seen as relatively low-throughput, the BiFC technique has so far been used to visualise more than two hundred protein–protein interactions. Finally, Petsalaki and Russell discuss protein interactions from a structural perspective. They describe how peptide-mediated interactions may play a larger role in cellular processes than previously thought and highlight the importance of computational methods which complement and accelerate the discovery and characterisation of protein–peptide interactions. Although it has long been assumed that the function of proteins can be reasonably well understood by considering the sum of its discrete structural units (functional domains), there is now mounting evidence that unfolded or intrinsically disordered regions within proteins can also determine a huge number of interactions that are biologically relevant. The detection of these lower affinity and transient protein–peptide interactions will require improvements in protein interaction screening technologies (such as those described in the following reviews), though may be more amenable to disruption by small molecules for the development of targeted therapies in disease. The authors propose that projects to systematically uncover the protein–peptide repertoire used in Nature will have numerous applications in molecular biology and medicine. Whilst not covered in this protein technologies section, peptide www.sciencedirect.com
Editorial overview Timms 315
arrays hold great promise for studying the determinants of protein–peptide interactions, although technical difficulties in the large-scale expression and purification of recombinant proteins that are stable and properly folded must be overcome. In summary, this section describes new and developing methods for studying protein interactions, multi-protein
www.sciencedirect.com
complexes and protein networks. This ‘interactome’ work has so far provided tremendous insight into protein function and the molecular mechanisms by which cellular processes are orchestrated. As the focus of molecular biology research shifts more towards the characterisation of gene products, it will depend increasingly on these technologies.
Current Opinion in Biotechnology 2008, 19:313–315
Protein technologies Editorial overview John F Timms Current Opinion in Biotechnology 2008, 19:313–315 Available online 11th August 2008 0958-1669/$ – see front matter # 2008 Elsevier Ltd. All rights reserved. DOI 10.1016/j.copbio.2008.07.003
John F Timms Cancer Proteomics Laboratory, EGA Institute for Women’s Health, Cruciform Building, University College London, London, UK e-mail: [email protected]
John F Timms, DPhil, is a senior scientist at the EGA Institute for Women’s Health, University College London where he is head of the Cancer Proteomics Laboratory. He has worked at Harvard Medical School and the Ludwig Institute for Cancer Research before taking up his current post at University College London. His research activities are focused on understanding the molecular mechanisms of growth factor and stress-activated signalling in normal and cancer cells and the discovery of cancer biomarkers using proteomics-based approaches and other techniques.
Proteins are the functional representations of the genes which encode them and they regulate and perform almost all biological processes in cells and tissues. Since nearly all coding genes have now been identified using genomic methods, our next challenge is to define the molecular and biological functions of these gene products. This is a daunting task given the unique nature of every protein and all the possible modifications that they are subject to. For this reason, novel post-genomic technologies are required to interrogate protein function on a large scale. An integral part of the functional activity of proteins revolves around their specific interactions with one another. Identifying protein–protein interactions, particularly in the context of different cellular processes or responses, is therefore critical to understanding protein function and hence cell biology. Protein–protein interactions also play a major role in disease and there is great promise in the development of therapeutic agents which can specifically target protein– protein interactions. This section reviews the different technologies that are used for studying protein interactions both in vitro and in vivo and which can be applied on a large, even genome-wide, scale. The reviews include recent methodological advances which are allowing researchers to now define weak or transient protein–protein interactions and the interactions of membrane proteins which have previously been difficult to study due to their nature. Applications of these methodologies in specific areas of cell biology are highlighted, including the study of cellular signalling events and the identification of dynamic changes in protein complexes using quantitative methods. The reviews cover reporter-based systems such as the yeast twohybrid (YTH) technology used for large-scale screening of protein interactions, through to more targeted epitope-tagging, purification and mass spectrometry (MS)-based approaches for characterising the components of multi-protein complexes. The principles and applications of resonance energy transfer (RET) methods for studying specific interactions in vivo is also covered. The last review discusses our current knowledge of structural determinants of protein–protein interactions and tools used for predicting protein interactions in silico. The latter also includes reference to useful resources of protein interaction data and software for interrogating these databases. Finally, the reviews provide a valuable resource for further reading and cover the seminal papers that have contributed to each area of this emerging and exciting field. An important starting point for understanding any protein’s function is knowledge of all possible interaction partners for that protein. In this respect, genome-wide YTH technology can be applied relatively easily with the availability of genomic or cDNA prey libraries expressing all
www.sciencedirect.com
Current Opinion in Biotechnology 2008, 19:313–315
314 Systems biology
encoded or expressed proteins in the organism or tissue of interest. In their review, Suter, Kittanakom and Stagljar provide a thorough account of the basic YTH technology and its many variants. These variants now allow interaction screening in a context that better reflects the physiological setting where the interactions occur. For example, adaptations for studying membrane protein interactions and use in mammalian cell systems are discussed. The review also provides a background on the pitfalls of the technology and how it can be adapted to decipher signalling pathways, to study the cellular compartmentalisation of interactions, to characterise interfaces within protein complexes and for the screening of small molecules with potential therapeutic use. A major drawback of the basic YTH technology is the relatively high number of false negatives and positives found in screens. These arise due to a failure of nuclear localization, improper folding of fusion proteins, a lack of cofactors necessary for interactions, forced overexpression of heterologous proteins and self-activation of the reporter gene by the bait. To partly overcome this problem, Collins and Choudhary outline how various affinity purification strategies can be combined with tandem MS for the identification of protein–protein interactions in more appropriate cellular models. In particular, it describes the design and use of single epitope and tandem affinity purification (TAP) tags, thereby bypassing the difficulties of generating antibodies against each target protein of interest. It discusses the optimisation of purification conditions, experimental design and the data analysis techniques which have been used to improve the sensitivity and specificity of detecting bona fide protein–protein interactions in different model systems. The authors describe seminal work where through the advent of high accuracy and high throughput MS instrumentation it is now possible to perform near proteome-wide analyses of affinity-purified multi-protein complexes, and when combined with MS-based quantification methods such as SILAC, it has become possible to study the dynamics of protein–protein interactions on a large scale. The authors also discuss recently developed methods for the identification of transient protein–protein interactions and introduce the subject of discriminating specific from non-specific binders. The problem of specificity and the detection of weak or transient interactions using affinity purification and MSbased approaches are further covered in the review by Vermeulen, Hubner and Mann. Whilst extensive purification is required to identify specific interactions, this comes at the cost of reduced throughput and the potential failure in detecting the weak interactors in protein complexes. After reviewing state-of-the-art MS-based quantification methods, the authors describe recently developed stable isotope-based quantitative proteomic methods which are being used to directly compare specific and Current Opinion in Biotechnology 2008, 19:313–315
control (single-step) pull downs. Specific interactors are revealed by their quantitative ratios (heavy versus light tag) and can be retrieved from the excess of non-specific background proteins. However, the authors warn that exchange of dynamic interaction partners can occur during incubation of mixed heavy and light-labelled samples and therefore must be controlled. They also describe recent work where mass tagging has been combined with RNAi for the generation of control samples to study endogenous protein complexes by immunoprecipitation, rather than using overexpressed epitope-tagged proteins and non-immune or ‘bead-only’ controls. Studies using isotopically labelled cross-linkers are also described where the aim is to stabilise and quantify multi-protein complexes. The application of these methods has proven successful in the identification of novel and post-translational modification-specific protein complexes in tyrosine kinase signalling, cell adhesion and chromatin biology. The review by Ciruela next focuses on non-invasive fluorescence-based methods using resonance energy transfer (RET). These technologies, namely Bioluminescence-RET(BRET) and Fluorescence-RET (FRET), and those centred on protein fragment complementation, such as Bimolecular Fluorescence Complementation (BiFC), have been successfully applied for visualising interactions occurring within protein complexes in living cells. The individual strengths and weaknesses of these methods when applied to the study of protein–protein interactions are discussed. Although these methods are seen as relatively low-throughput, the BiFC technique has so far been used to visualise more than two hundred protein–protein interactions. Finally, Petsalaki and Russell discuss protein interactions from a structural perspective. They describe how peptide-mediated interactions may play a larger role in cellular processes than previously thought and highlight the importance of computational methods which complement and accelerate the discovery and characterisation of protein–peptide interactions. Although it has long been assumed that the function of proteins can be reasonably well understood by considering the sum of its discrete structural units (functional domains), there is now mounting evidence that unfolded or intrinsically disordered regions within proteins can also determine a huge number of interactions that are biologically relevant. The detection of these lower affinity and transient protein–peptide interactions will require improvements in protein interaction screening technologies (such as those described in the following reviews), though may be more amenable to disruption by small molecules for the development of targeted therapies in disease. The authors propose that projects to systematically uncover the protein–peptide repertoire used in Nature will have numerous applications in molecular biology and medicine. Whilst not covered in this protein technologies section, peptide www.sciencedirect.com
Editorial overview Timms 315
arrays hold great promise for studying the determinants of protein–peptide interactions, although technical difficulties in the large-scale expression and purification of recombinant proteins that are stable and properly folded must be overcome. In summary, this section describes new and developing methods for studying protein interactions, multi-protein
www.sciencedirect.com
complexes and protein networks. This ‘interactome’ work has so far provided tremendous insight into protein function and the molecular mechanisms by which cellular processes are orchestrated. As the focus of molecular biology research shifts more towards the characterisation of gene products, it will depend increasingly on these technologies.
Current Opinion in Biotechnology 2008, 19:313–315