‘Unknown’ proteins and ‘orphan’ enzymes: the missing half of the engineering parts list – and how to find it

Hanson, Andrew D.; Pribat, Anne; Waller, Jeffrey C.; Crécy‐Lagard, Valérie de

doi:10.1042/bj20091328

Cited by 178 publications

(169 citation statements)

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“…While the combined use of sequence and (co)expression data can strengthen inferences (Mutwil et al, 2011), there remains much room for error. Indeed, there is an emerging suspicion that seemingly similar enzymes may often have fundamentally different roles, including scavenging of metabolites that are created due to infidelities in catalysis by other enzymes (Hanson et al, 2010). Another constraint affects the use of information about protein abundance to draw inferences about metabolic events.…”

mentioning

confidence: 99%

On the Discordance of Metabolomics with Proteomics and Transcriptomics: Coping with Increasing Complexity in Logic, Chemistry, and Network Interactions Scientific Correspondence

2012

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We suspect that many biologists who have been following the development of functional 'omics and systems biology are wondering why, compared with the success in integrating information from large-scale studies of transcripts and, more recently, proteins, there is less success in integrating information about metabolism.Information about thousands of transcripts has been integrated into large databases that are used by thousands of scientists (Zimmermann et al., 2004). Rapid advances will soon allow a similar approach in quantitative proteomics. Strategies also exist to integrate information about the expression of genes, as transcripts, with information about changes in the levels of the encoded proteins. Even when changes in transcript levels do not lead to changes in the levels of the encoded proteins, this apparent discrepancy can often be explained by quantitative analysis of the data sets and information about translational regulation and protein turnover (Piques et al., 2009; Schwanhäusser et al., 2011). Such advances are providing systemslevel information about transcriptional and signaling networks that regulate the transcript and protein abundance. However, another important goal of systems biology is a comprehensive understanding of the functional importance of these changes in transcript and protein abundance. Although there is sometimes an encouragingly simple relationship between changes of transcripts and proteins and changes in downstream biological functions, quite often there is a major discordance. In this scientific correspondence, we will discuss reasons for this apparent disconnect, which lie partly in technical issues and partly in the complexity and structure of metabolic networks, and then propose some priorities in metabolic research to combat these problems.As we climb up what has been described as the "pyramid of life" (Barabási and Oltvai, 2004) from gene through RNA and protein to phenotype (which includes chemical composition), we are confronted with an increasing complexity both at the chemical level and in the logic that is needed to draw inferences from one level to the next. Nucleic acids consist of four bases, joined by the same phosphoester bond and with pairwise interacting side chains. This simple structure allows a near-to-binary storage and transmission of information. Proteins are chemically much more complex, due to the functionalities of 20 different amino acid side chains. This large expansion in chemical complexity lies at the center of the flexibility and complexity of biological systems, as it allows different proteins to perform an enormous range of different functions. Nevertheless, proteins still have a simple primary structure, with amino acids linked via a peptide bond in a fixed sequence that, via the universal genetic code, can be unambiguously linked to the sequence of nucleic acids (Aebersold and Mann, 2003;Fernie et al., 2004; Baerenfaller et al., 2008). This logic breaks down when we consider entities that are generated by the activities of individual pr...

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confidence: 99%

On the Discordance of Metabolomics with Proteomics and Transcriptomics: Coping with Increasing Complexity in Logic, Chemistry, and Network Interactions Scientific Correspondence

2012

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“…Taken together, our results pave the way for the large-scale generation of endogenously tagged human cell lines for the proteome-wide analysis of protein localization and interaction networks in a native cellular context. CRISPR/Cas9 | GFP library | genome engineering M ore than a decade after the completion of the Human Genome Project (1), over 30% of human genes still lack clear functional annotation (2,3). Functional tagging is a powerful strategy to characterize the cellular role of proteins.…”

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confidence: 99%

A scalable strategy for high-throughput GFP tagging of endogenous human proteins

Leonetti

Sekine

Kamiyama

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

229

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A central challenge of the postgenomic era is to comprehensively characterize the cellular role of the ∼20,000 proteins encoded in the human genome. To systematically study protein function in a native cellular background, libraries of human cell lines expressing proteins tagged with a functional sequence at their endogenous loci would be very valuable. Here, using electroporation of Cas9 nuclease/single-guide RNA ribonucleoproteins and taking advantage of a split-GFP system, we describe a scalable method for the robust, scarless, and specific tagging of endogenous human genes with GFP. Our approach requires no molecular cloning and allows a large number of cell lines to be processed in parallel. We demonstrate the scalability of our method by targeting 48 human genes and show that the resulting GFP fluorescence correlates with protein expression levels. We next present how our protocols can be easily adapted for the tagging of a given target with GFP repeats, critically enabling the study of low-abundance proteins. Finally, we show that our GFP tagging approach allows the biochemical isolation of native protein complexes for proteomic studies. Taken together, our results pave the way for the large-scale generation of endogenously tagged human cell lines for the proteome-wide analysis of protein localization and interaction networks in a native cellular context. CRISPR/Cas9 | GFP library | genome engineering M ore than a decade after the completion of the Human Genome Project (1), over 30% of human genes still lack clear functional annotation (2, 3). Functional tagging is a powerful strategy to characterize the cellular role of proteins. In particular, tags allow access to two key features of protein function: localization (using fluorescent tags) and interaction partners (using epitope tags and immunoprecipitation). Hence, by tagging proteins in a systematic manner, a comprehensive functional description of an organism's proteome can be achieved. The power of systematic tagging approaches is best illustrated by studies conducted in the budding yeast Saccharomyces cerevisiae (4). In particular, a genome-wide collection of GFP-tagged yeast strains enabled the systematic study of protein localization in live cells (5), whereas libraries of strains expressing TAP epitope-fusion proteins paved the way for the large-scale isolation and proteomic analysis of protein complexes (6, 7). One of the great advantages of yeast genetics (especially in S. cerevisiae) is the efficiency and relative simplicity of PCR-based homologous recombination (8). As a result, functional tags can be easily inserted in a gene locus of interest, preserving endogenous expression levels and minimizing genomic disruption. Together, these genome-wide tagged libraries helped provide a comprehensive snapshot of the yeast protein landscape under near-native conditions (4, 5, 9-11).The development of clustered regularly interspersed short palindromic repeat associated protein 9 (CRISPR/Cas9)-based methods has profoundly transformed our ability to dir...

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“…where there is either a completely different enzyme (nonorthologous displacement) or an alternative pathway (16,17). Here, we report an investigation of these possibilities using comparative genomics analysis.…”

Section: -Formyltetrahydrofolate (5-cho-thf)mentioning

confidence: 99%

Moonlighting Glutamate Formiminotransferases Can Functionally Replace 5-Formyltetrahydrofolate Cycloligase

Jeanguenin

Lara‐Núñez

Pribat

et al. 2010

Journal of Biological Chemistry

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‘Unknown’ proteins and ‘orphan’ enzymes: the missing half of the engineering parts list – and how to find it

Cited by 178 publications

References 93 publications

On the Discordance of Metabolomics with Proteomics and Transcriptomics: Coping with Increasing Complexity in Logic, Chemistry, and Network Interactions Scientific Correspondence

On the Discordance of Metabolomics with Proteomics and Transcriptomics: Coping with Increasing Complexity in Logic, Chemistry, and Network Interactions Scientific Correspondence

A scalable strategy for high-throughput GFP tagging of endogenous human proteins

Moonlighting Glutamate Formiminotransferases Can Functionally Replace 5-Formyltetrahydrofolate Cycloligase

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