Pitter F. Huesgen

Proteome-wide analysis of protein carboxy termini: C terminomics

As proteome-wide C-terminal sequence analysis has been largely intractable, we developed a polymer-based enrichment approach to profile protein C-terminal peptides by mass spectrometry and identified hundreds of C-terminal peptides in the Escherichia coli proteome. We isotopically labeled GluC protease–digested and undigested samples and identified GluC substrates and their cleavage sites by quantification of neo–C-terminal peptides. Our method thus enables global annotation of protein C-terminal posttranslational modifications, including proteolytic truncations.

Dual specificities of the glyoxysomal/peroxisomal processing protease Deg15 in higher plants.

Glyoxysomes are a subclass of peroxisomes involved in lipid mobilization. Two distinct peroxisomal targeting signals (PTSs), the C-terminal PTS1 and the N-terminal PTS2, are defined. Processing of the PTS2 on protein import is conserved in higher eukaryotes. The cleavage site typically contains a Cys at P1 or P2. We purified the glyoxysomal processing protease (GPP) from the fat-storing cotyledons of watermelon (Citrullus vulgaris) by column chromatography, preparative native isoelectric focusing, and 2D PAGE. The GPP appears in two forms, a 72-kDa monomer and a 144-kDa dimer, which are in equilibrium with one another. The equilibrium is shifted on Ca2+ removal toward the monomer and on Ca2+ addition toward the dimer. The monomer is a general degrading protease and is activated by denatured proteins. The dimer constitutes the processing protease because the substrate specificity proven for the monomer (Φ-Arg/Lys↓) is different from the processing substrate specificity (Cys-Xxx↓/Xxx-Cys↓) found with the mixture of monomer and dimer. The Arabidopsis genome analysis disclosed three proteases predicted to be in peroxisomes, a Deg-protease, a pitrilysin-like metallopeptidase, and a Lon-protease. Specific antibodies against the peroxisomal Deg-protease from Arabidopsis (Deg15) identify the watermelon GPP as a Deg15. A knockout mutation in the DEG15 gene of Arabidopsis (At1g28320) prevents processing of the glyoxysomal malate dehydrogenase precursor to the mature form. Thus, the GPP/Deg15 belongs to a group of trypsin-like serine proteases with Escherichia coli DegP as a prototype. Nevertheless, the GPP/Deg15 possesses specific characteristics and is therefore a new subgroup within the Deg proteases.

Characterization of the prime and non-prime active site specificities of proteases by proteome-derived peptide libraries and tandem mass spectrometry

To link cleaved substrates in complex systems with a specific protease, the protease active site specificity is required. Proteomic identification of cleavage sites (PICS) simultaneously determines both the prime- and non-prime-side specificities of individual proteases through identification of hundreds of individual cleavage sequences from biologically relevant, proteome-derived peptide libraries. PICS also identifies subsite cooperativity. To generate PICS peptide libraries, cellular proteomes are digested with a specific protease such as trypsin. Following protease inactivation, primary amines are protected. After incubation with a test protease, each prime-side cleavage fragment has a free newly formed N-terminus, which is biotinylated for affinity isolation and identification by liquid chromatography–tandem mass spectrometry. The corresponding non-prime sequences are derived bioinformatically. The step-by-step protocol also presents a web service for PICS data analysis, as well as introducing and validating PICS peptide libraries made from Escherichia coli.

The DEG15 Serine Protease Cleaves Peroxisomal Targeting Signal 2-Containing Proteins in Arabidopsis

Two distinct peroxisomal targeting signals (PTSs), the C-terminal PTS1 and the N-terminal PTS2, are defined. Processing of the PTS2 on protein import is conserved in higher eukaryotes. Recently, candidates for the responsible processing protease were identified from plants (DEG15) and mammals (TYSND1). We demonstrate that plants lacking DEG15 show an expressed phenotype potentially linked to reduced β-oxidation, indicating the impact of protein processing on peroxisomal functions in higher eukaryotes. Mutational analysis of Arabidopsis (Arabidopsis thaliana) DEG15 revealed that conserved histidine, aspartic acid, and serine residues are essential for the proteolytic activity of this enzyme in vitro. This indicates that DEG15 and related enzymes are trypsin-like serine endopeptidases. Deletion of a plant-specific stretch present in the protease domain diminished, but did not abolish, the proteolytic activity of DEG15 against the PTS2-containing glyoxysomal malate dehydrogenase. Fluorescence microscopy showed that a DEG15-green fluorescent protein fusion construct is targeted to peroxisomes in planta. In vivo studies with isolated homozygous deg15 knockout mutants and complemented mutant lines suggest that this enzyme mediates general processing of PTS2-containing proteins.

Annotating N Termini for the Human Proteome Project: N Termini and Nα-Acetylation Status Differentiate Stable Cleaved Protein Species from Degradation Remnants in the Human Erythrocyte Proteome

A goal of the Chromosome-centric Human Proteome Project is to identify all human protein species. With 3844 proteins annotated as “missing”, this is challenging. Moreover, proteolytic processing generates new protein species with characteristic neo-N termini that are frequently accompanied by altered half-lives, function, interactions, and location. Enucleated and largely void of internal membranes and organelles, erythrocytes are simple yet proteomically challenging cells due to the high hemoglobin content and wide dynamic range of protein concentrations that impedes protein identification. Using the N-terminomics procedure TAILS, we identified 1369 human erythrocyte natural and neo-N-termini and 1234 proteins. Multiple semitryptic N-terminal peptides exhibited improved mass spectrometric identification properties versus the intact tryptic peptide enabling identification of 281 novel erythrocyte proteins and six missing proteins identified for the first time in the human proteome. With an improved bioinformatics workflow, we developed a new classification system and the Terminus Cluster Score. Thereby we described a new stabilizing N-end rule for processed protein termini, which discriminates novel protein species from degradation remnants, and identified protein domain hot spots susceptible to cleavage. Strikingly, 68% of the N-termini were within genome-encoded protein sequences, revealing alternative translation initiation sites, pervasive endoproteolytic processing, and stabilization of protein fragments in vivo. The mass spectrometry proteomics data have been deposited to ProteomeXchange with the data set identifier .

TopFIND 2.0—linking protein termini with proteolytic processing and modifications altering protein function

Protein termini provide critical insights into the functional state of individual proteins. With recent advances in specific proteomics approaches to enrich for N- and C-terminomes, the global analysis of whole terminomes at a proteome-wide scale is now possible. Information on the actual N- and C-termini of proteins in vivo and any post-translational modifications, including their generation by proteolytic processing, is rapidly accumulating. To access this information we present version 2.0 of TopFIND (http://clipserve.clip.ubc.ca/topfind), a knowledgebase for protein termini, terminus modifications and underlying proteolytic processing. Built on a protein-centric framework TopFIND covers five species: Homo sapiens, Mus musculus, Arabidopsis thaliana, Saccharomyces cerevisiae and Escherichia coli and incorporates information from curated community submissions, publications, UniProtKB and MEROPS. Emphasis is placed on the detailed description and classification of evidence supporting the reported identification of each cleavage site, terminus and modification. A suite of filters can be applied to select supporting evidence. A dynamic network representation of the relationship between proteases, their substrates and inhibitors as well as visualization of protease cleavage site specificities complements the information displayed. Hence, TopFIND supports in depth investigation of protein termini information to spark new hypotheses on protein function by correlating cleavage events and termini with protein domains and mutations.

LysargiNase mirrors trypsin for protein C-terminal and methylation-site identification

To improve proteome coverage and protein C-terminal identification, we characterized the Methanosarcina acetivorans thermophilic proteinase LysargiNase, which cleaves before lysine and arginine up to 55 °C. Unlike trypsin, LysargiNase-generated peptides had N-terminal lysine or arginine residues and fragmented with b ion–dominated spectra. This improved protein C terminal–peptide identification and several arginine-rich phosphosite assignments. Notably, cleavage also occurred at methylated or dimethylated lysine and arginine, facilitating detection of these epigenetic modifications.

N- and C-terminal degradomics: new approaches to reveal biological roles for plant proteases from substrate identification.

Proteolysis is an irreversible post-translational modification that regulates many intra- and intercellular processes, including essential go/no-go decisions during cell proliferation, development and cell death. Hundreds of protease-coding genes have been identified in plants, but few have been linked to specific substrates. Conversely, proteolytic processes are frequently observed in plant biology but rarely have they been ascribed to specific proteases. In mammalian systems, unbiased system-wide proteomics analyses of protease activities have recently been tremendously successful in the identification of protease substrate repertoires, also known as substrate degradomes. Knowledge of the substrate degradome is key to understand the role of proteases in vivo. Quantitative shotgun proteomic studies have been successful in identifying protease substrates, but while simple to perform they are biased toward abundant proteins and do not reveal precise cleavage sites. Current degradomics techniques overcome these limitations by focusing on the information-rich amino- and carboxy-terminal peptides of the original mature proteins and the protease-generated neo-termini. Targeted quantitative analysis of protein termini identifies precise cleavage sites in protease substrates with exquisite sensitivity and dynamic range in in vitro and in vivo systems. This review provides an overview of state-of-the-art methods for enrichment of protein terminal peptides, and their application to protease research. These emerging degradomics techniques promise to clarify the elusive biological roles of proteases and proteolysis in plants.

Proteolysis of cystatin C by cathepsin D in the breast cancer microenvironment

The aspartic protease cathepsin D, a poor prognostic indicator of breast cancer, is abundantly secreted as procathepsin D by human breast cancer cells and self‐activates at low pH in vitro, giving rise to catalytically active cathepsin D. Due to a lower extracellular pH in tumor microenvironments compared to normal tissues, cathepsin D may cleave pathophysiological substrates contributing to cancer progression. Here, we show by yeast 2‐hybrid and degradomics analyses that cystatin C, the most potent natural secreted inhibitor of cysteine cathepsins, both binds to and is a substrate of extracellular procathepsin D. The amount of cystatin C in the extracellular environment is reduced in the secretome of mouse embryonic fibroblasts stably transfected with human cathepsin D. Cathepsin D extensively cleaved cystatin C in vitro at low pH. Cathepsin D secreted by breast cancer cells also processed cystatin C at the pericellular pH of tumors and so enhancing extracellular proteolytic activity of cysteine cathepsins. Thus, tumor derived cathepsin D assists breast cancer progression by reducing cystatin C activity, which, in turn, enhances cysteine cathepsin proteolytic activity, revealing a new link between protease classes in the protease web.—Laurent‐Matha, V., Huesgen, P. F., Masson, O., Derocq, D., Prébois, C., Gary‐Bobo, M., Lecaille, F., Rebière, B., Meurice, G., Oréar, C., Hollingsworth, R. E., Abrahamson, M., Lalmanach, G., Overall, C. M., Liaudet‐Coopman, E. Proteolysis of cystatin C by cathepsin D in the breast cancer microenvironment. FASEB J. 26, 5172–5181 (2012). www.fasebj.org

Proteomic protease specificity profiling of clostridial collagenases reveals their intrinsic nature as dedicated degraders of collagen

Clostridial collagenases are among the most efficient degraders of collagen. Most clostridia are saprophytes and secrete proteases to utilize proteins in their environment as carbon sources; during anaerobic infections, collagenases play a crucial role in host colonization. Several medical and biotechnological applications have emerged utilizing their high collagenolytic efficiency. However, the contribution of the functionally most important peptidase domain to substrate specificity remains unresolved. We investigated the active site sequence specificity of the peptidase domains of collagenase G and H from Clostridium histolyticum and collagenase T from Clostridium tetani. Both prime and non-prime cleavage site specificity were simultaneously profiled using Proteomic Identification of protease Cleavage Sites (PICS), a mass spectrometry-based method utilizing database searchable proteome-derived peptide libraries. For each enzyme we identified > 100 unique-cleaved peptides, resulting in robust cleavage logos revealing collagen-like specificity patterns: a strong preference for glycine in P3 and P1′, proline at P2 and P2′, and a slightly looser specificity at P1, which in collagen is typically occupied by hydroxyproline. This specificity for the classic collagen motifs Gly-Pro-X and Gly-X-Hyp represents a remarkable adaptation considering the complex requirements for substrate unfolding and presentation that need to be fulfilled before a single collagen strand becomes accessible for cleavage. Biological significance We demonstrate the striking sequence specificity of a family of clostridial collagenases using proteome derived peptide libraries and PICS, Proteomic Identification of protease Cleavage Sites. In combination with the previously published crystal structures of these proteases, our results represent an important piece of the puzzle in understanding the complex mechanism underlying collagen hydrolysis, and pave the way for the rational design of specific test substrates and selective inhibitors. This article is part of a Special Issue entitled: Can Proteomics Fill the Gap Between Genomics and Phenotypes?