Degradomics

Degradomics is a sub-discipline of biology encompassing all the genomic and proteomic approaches devoted to the study of proteases, their inhibitors, and their substrates on a system-wide scale.McQuibban, G.A., Gong J., Tam E.M., McCulloch C.A.G., Clark-Lewis I., Overall C.M.. Inflammation dampened by gelatinase A cleavage of monocyte chemoattractant protein-3. Science 289, 1202–1206 (2000).López-Otin, C., Overall, C.M. Protease degradomics: a new challenge for proteomics. Nat. Rev. Mol. Cell Biol. 3, 509–519 (2002). This includes the analysis of the protease and protease-substrate repertoires, also called "protease degradomes".Overall C M., Blobel C. P. In search of partners: linking extracellular proteases to substrates. Nat. Rev. Mol. Cell Biol. 8, 245–257 (2007). The scope of these degradomes can range from cell, tissue, and organism-wide scales.

Background

As the second largest class of enzymes behind ubiquitin ligases and responsible for ~2% of any organism's genes,Puente X. S., Sánchez L. M., Overall C. M., López-Otín C. Human and mouse proteases: a comparative genomic approach. Nature Reviews Genetics. 4(7):544–558 (2003). proteases have drawn the attention of biologists to develop a field aimed at identifying and quantifying their roles in biology. First coined in 2000 by the Overall Lab in McQuibban et al., degradomics was described as linking proteases to substrates on a proteome basis. The discoveries of novel roles for proteases and breakthroughs in protease-substrate discovery would be summarized later by Dr. Carlos Lopez-Otin and Dr. Chris Overall, introducing degradomics on a system-wide scale. They collated the current and emerging techniques available to describe proteolysis. By drawing attention to how proteolysis serves as an additional irreversible mechanism by which cells could achieve control over biological processes, they outlined the necessity of studying proteases for their functional relevance in processing bioactive molecules. These bioactive molecules play roles in coagulation, complement activation, DNA replication, cell-cycle control, cellular proliferation and migration, hemostasis, immunity, and apoptosis.Barrett, A. J. et al. (eds) Handbook of Proteolytic Enzymes (Academic, London, 1998).Sternlicht, M. D., Werb, Z. How matrix metalloproteinases regulate cell behavior. Annu. Rev. Cell Dev. Biol. 17, 463–516 (2001). The degradome was broken down into two concepts, the first referring the entire profile of proteases expressed under by a cell, tissue, or organism under defined circumstances. The second definition applies specifically to the full substrate repertoire of a certain protease in a cell, tissue, or organism.

Dr. Overall's group would go on to annotate the complete human and mouse protease-inhibitor degradomes in 2003. As the complexity of proteolytic networks was uncovered, more thorough descriptions of the degradome and ways to study it became necessary. In 2007, Dr. Overall updated the field with a review co-authored by Dr. Carl Blobel detailing how advanced methods were revolutionizing protease-substrate discovery. They described the process of linking proteases to their substrates on a step by step process, beginning with biochemical and proteomic discovery, validation using cellular based assays, and progressing to whole organism levels using animal models. More recently, as technology and techniques have advanced, the Overall Lab and others have continued to direct the field using more powerful and quantitative techniques.Marino G., Eckhard U., Overall C. M. Protein Termini and Their Modifications Revealed by Positional Proteomics. ACS Chem. Biol. 10, 1754-1764 (2015).Vizovisek M., Vidmar R., Fonovic M., Turk B. Current trends and challenges in proteomic identification of protease substrates. Biochimie. 122, 77-87 (2016).

Methods

Transcriptomic Methods

Gene Microarrays

Traditionally DNA microarrays use complementary DNA or oligonucleotide probes to analyze messenger RNA (mRNA) from genes of interest. Extracted total RNA serves as a template for complementary DNA (cDNA) that is tagged with fluorescent probes before being allowed to hybridize to the microarray for visualization.Yang YH, Speed T. Design issues for cDNA microarray experiments. Nat. Rev. Genet. 3, 579–588 (2002) For proteases, specific probes for protease genes and their inhibitors have been developed to view expression patterns on the mRNA transcript level. The two platforms currently available for this purpose come from corporate and academic sources. Affymetrix's Hu/Mu ProtIn Microarray uses 516 and 456 probe sets to evaluate human and murine proteases, inhibitors, and interactors respectively.Schwartz D.R., Moin K., Yao B., Matrisian L.M., Coussens L.M., Bugge T.H., Fingleton B., Acuff H.B., Sinnamon M., Nassar H., Platts A.E., Krawetz S.A., Linebaugh B.E., Sloane B.F. Hu/Mu ProtIn oligonucleotide microarray: dual-species array for profiling protease and protease inhibitor gene expression in tumors and their microenvironment. Mol. Cancer Res., 5 (5) 443–454 (2007).Acuff H.B., Sinnamon M., Fingleton B., Boone B., Levy S.E., Chen X., Pozzi A., Carbone D.P., Schwartz D.R., Moin K., Sloane B.F., Matrisian L.M. Analysis of host- and tumor-derived proteinases using a custom dual species microarray reveals a protective role for stromal matrix metalloproteinase-12 in non-small cell lung cancer Cancer Res., 66 (16) 7968–7975 (2006). CLIP-CHIP™, developed by the Overall Lab, is a complete protease and inhibitor DNA microarray for all 1561 human and murine proteases, non-proteolytic homologues, and their inhibitors.Overall C. M., Tam E.M., Kappelhoff R., Connor A., Ewart T., Morrison C.J., Puente X., Lopez-Otin C., Seth A. Protease degradomics: mass spectrometry discovery of protease substrates and the CLIP-CHIP, a dedicated DNA microarray of all human proteases and inhibitors. Biol. Chem. 385, 493–504 (2004).Kappelhoff R., Auf dem Keller U., Overall C.M. Analysis of the degradome with the CLIP-CHIP microarray. Methods Mol. Biol.622:175-93 (2010).Kappelhoff R., Overall C.M. The CLIP-CHIP oligonucleotide microarray: dedicated array for analysis of all protease, nonproteolytic homolog, and inhibitor gene transcripts in human and mouse. Curr. Protoc. Protein Sci. 2007 Aug; Chapter 21:Unit 21.19. Both of these tools allow comparison of expression patterns between normal and diseased samples and tissues. Unfortunately, as transcript levels often fail to reflect protein expression levels, gene microarrays are limited in representing protein in samples.Lundberg E., Fagerberg L., Klevebring D., Matic I., Geiger T., Cox J., Algenas C., Lundeberg J., Mann M., Uhlen M. Defining the transcriptome and proteome in three functionally different human cell lines. Mol. Syst. Biol. 6:450 (2010). In addition, proteases recruited from remote sources like nearby tissues are ignored by these DNA based arrays, reiterating the need for protein based methods to confirm the presence and activity of functional enzymes when transcriptome analysis is performed.

Quantitative real-time polymerase chain reaction (qRT-PCR)

A more sensitive approach to transcript analysis of a gene is quantitative real-time polymerase chain reaction (qRT-PCR), which has also seen action in quantifying protease mRNA levels. Again, total RNA is extracted and used to generate cDNA for PCR amplification. As long as there is a specific primer for a protease, protease inhibitor, or interacting molecule, qRT-PCR can serve as a highly sensitive method to detect minuscule amounts of mRNA copy numbers per cell.Nuttall R.K., Pennington C.J., Taplin J., Wheal A., Yong V.W., Forsyth P.A., Edwards D.R. Elevated membrane-type matrix metalloproteinases in gliomas revealed by profiling proteases and inhibitors in human cancer cells. Mol. Cancer Res. 1 (5) 333-345 (2003). A drawback that separates it from microarray analysis is its limited scope: a microarray can handle parallel analysis of multiple genes while qRT-PCR must amplify one mRNA for analysis at a time. It also suffers from the same limitation of microarray analysis regarding the lack of correlation between transcript and protein levels. However, its sensitivity lends it as a useful tool in validating microarray findings and quantifying specific protease transcripts of interest.

RNA sequencing (RNA-seq)

Whole transcriptome shotgun sequencing (WTSS)Morin R., Bainbridge M., Fejes A., Hirst M., Krzywinski M., Pugh T., McDonald H., Varhol R., Jones S., Marra M. Profiling the HeLa S3 transcriptome using randomly primed cDNA and massively parallel short-read sequencing. Biotechniques 45 (1):81-94 (2008). is the latest in gene expression studies, using next generation sequencing (NGS) to quantify RNA in samples on a high throughput scale.Wang Z., Gerstein M., Snyder M. RNA-Seq: a revolutionary tool for transcriptomics. Nat. Rev. Genet. 10(1):57-63 (2009). As biology trends toward using RNA-seq over microarray analysis in evaluating the transcriptome, so does degradomics. The field adapts the approach to analyzing the presence and quantity of transcripts of proteases, their substrates, and their inhibitors. While developed microarrays remain a major workhorse in studying gene expression in degradomics, its limitations of cross hybridization and dynamic range issues suggest RNA-seq will take a larger role as costs decrease and analysis improves.

Genomic Methods

Yeast Two-hybrid Screens

Yeast two-hybrid analyses have been adapted for protease-substrate discovery. As protease exosites play roles in protein-protein recognition and interaction, biologists have used exosites as tools to screen for protease interactors and potential substrates. These protease exosite scanning assays use protease exosites as bait to scan a cDNA library for possible interacting partners.

Another early adaptation of yeast two-hybrid screening in protease-substrate discovery is Inactive-catalytic-domain capture (ICDC).Overall C. M., Tam E. M., Kappelhoff R., Connor A., Ewart T., Morrison C. J., Puente X., López-Otín C., and Seth A. Protease degradomics: mass spectrometry discovery of protease substrates and the CLIP-CHIP, a dedicated DNA microarray of all human proteases and inhibitors. Biol. Chem. 385, 493– 504 (2004). This approach attempts to avoid the limitation of protease exosite scanning, which fails to account for any substrates that do not require exosites to for recognition before cleavage. The bait for these assays are immobilized catalytically inactive mutant protease domains that cannot cleave and release their substrates once bound.

While useful in early degradomic studies, the limitations of adapted yeast two-hybrid screens have forced the field to move on to higher-throughput approaches for protease-substrate discovery. Their high rate of false positives and negatives,Uetz P., Giot L., Cagney G., Mansfield T. A., Judson R. S., Knight J. R., Lockshon D., Narayan V., Srinivasan M., Pochart P., Qureshi-Emili A., Li Y., Godwin B., Conover D., Kalbfleisch T., Vijayadamodar G., Yang M., Johnston M., Fields S., and Rothberg J. M. A comprehensive analysis of protein-protein interactions in Saccharomyces cerevisiae. Nature 403,623– 627 (2000).Ito T., Chiba T., Ozawa R., Yoshida M., Hattori M., Sakaki Y. A comprehensive two-hybrid analysis to explore the yeast protein interactome. Proc. Natl. Acad. Sci. U.S.A. 98, 4569– 4574 (2001). inability to recognize complex interactions, lack of biologically compartmentalization, and failure to account for post-translational modifications necessary for protein-protein interactions hamper their usefulness.Doucet A., Overall C.M. Protease proteomics: revealing protease in vivo functions using systems biology approaches Mol. Aspects Med., 29, 339–358 (2008). Thus they have been largely replaced by proteomic methods as technology has improved.

Proteomic Methods

Protease-specific Arrays