The C-degron pathway eliminates mislocalized proteins and products of deubiquitinating enzymes.

Protein termini are determinants of protein stability. Proteins bearing degradation signals, or degrons, at their amino- or carboxyl-termini are eliminated by the N- or C-degron pathways, respectively. We aimed to elucidate the function of C-degron pathways and to unveil how normal proteomes are exempt from C-degron pathway-mediated destruction. Our data reveal that C-degron pathways remove mislocalized cellular proteins and cleavage products of deubiquitinating enzymes. Furthermore, the C-degron and N-degron pathways cooperate in protein removal. Proteome analysis revealed a shortfall in normal proteins targeted by C-degron pathways, but not of defective proteins, suggesting proteolysis-based immunity as a constraint for protein evolution/selection. Our work highlights the importance of protein termini for protein quality surveillance, and the relationship between the functional proteome and protein degradation pathways.

Engineered peptide ligases for cell signaling and bioconjugation.

Enzymes that catalyze peptide ligation are powerful tools for site-specific protein bioconjugation and the study of cellular signaling. Peptide ligases can be divided into two classes: proteases that have been engineered to favor peptide ligation, and protease-related enzymes with naturally evolved peptide ligation activity. Here, we provide a review of key natural peptide ligases and proteases engineered to favor peptide ligation activity. We cover the protein engineering approaches used to generate and improve these tools, along with recent biological applications, advantages, and limitations associated with each enzyme. Finally, we address future challenges and opportunities for further development of peptide ligases as tools for biological research.

Tying up loose ends: the N-degron and C-degron pathways of protein degradation.

Selective protein degradation by the ubiquitin-proteasome system (UPS) is thought to be governed primarily by the recognition of specific motifs - degrons - present in substrate proteins. The ends of proteins - the N- and C-termini - have unique properties, and an important subset of protein-protein interactions involve the recognition of free termini. The first degrons to be discovered were located at the extreme N-terminus of proteins, a finding which initiated the study of the N-degron (formerly N-end rule) pathways, but only in the last few years has it emerged that a diverse set of C-degron pathways target analogous degron motifs located at the extreme C-terminus of proteins. In this minireview we summarise the N-degron and C-degron pathways currently known to operate in human cells, focussing primarily on those that have been discovered in recent years. In each case we describe the cellular machinery responsible for terminal degron recognition, and then consider some of the functional roles of terminal degron pathways. Altogether, a broad spectrum of E3 ubiquitin ligases mediate the recognition of a diverse array of terminal degron motifs; these degradative pathways have the potential to influence a wide variety of cellular functions.

Positional proteomics for identification of secreted proteoforms released by site-specific processing of membrane proteins.

Proteolytic processing shapes cellular interactions with the environment. As a pathway of unconventional protein secretion, ectodomain shedding releases soluble proteoforms of membrane-anchored proteins. This can trigger subsequent cleavage within the membrane stub and the release of additional soluble fragments to intra- and extracellular environments. Distinct membrane-bound proteases, or sheddases, may cleave the same membrane proteins at different sites. Determination of these precise cleavage sites is important, as differently processed proteoforms may exhibit distinct physiological properties and execute antagonistic paracrine and endocrine signaling functions. Conventional quantitative proteomic approaches reliably identify shed proteoforms, but typically not their termini and are thus not able distinguish between functionally different proteoforms differing only by a few amino acids. Dedicated positional proteomics overcomes this challenge and enables proteome-wide identification of protein N- and C-termini. Here, we review positional proteomics techniques, summarize their application to ectodomain shedding and discuss current challenges and developments.

Protein Termini.

Since the first and the last residues of a protein have peculiar properties, unique amongst all residues, they have been analyzed repeatedly during the last decades. In this brief review, I try to summarize, besides the biochemical roles, the five features that have attracted most attention: (i) the Euclidean distance between the N- and C-termini and its relevance to protein folding, (ii) the reason why the termini are solvent exposed, (iii) the backbone conformation of the termini, (iv) the amino acid composition of the termini, and (v) the role of the termini in protein crystallization. Each of these five issues, which deserve attention nowadays thanks to the availability of massive amounts of data, is accompanied by my personal outlook of the research in the field.

Ensembles of protein termini and specific proteolytic signatures as candidate biomarkers of disease.

Early accurate diagnosis and personalized treatment are essential in order to treat complex or fatal diseases such as cancer and autoimmune, cardiovascular and neurodegenerative diseases. To realize this vision, new diagnostic and prognostic biomarkers are urgently required. MS-based proteomics is the most promising approach for protein biomarker identification, but suffers in clinical translation of biomarker candidates that show only quantitative differences from normal tissue. Indeed, success in translating proteomic data to biomarkers in the clinic has been disappointing. Here, we propose that protein termini provide a new opportunity for biomarker discovery due to qualitative differences in intact and new protein termini between diseased and normal tissues. Altered proteolysis occurs in most pathologies. Disease- and process-specific protein modifications, including proteolytic processing and subsequent modification of the terminal amino acids, frequently lead to altered protein activity that plays key roles in the disease process. Thus, mapping of ensembles of characteristic protein termini provides a proteolytic signature of high information content that shows both quantitative and most importantly qualitative differences in different diseases and stage of disease. These unique protein biomarkers have the added benefit of being mechanistically informative by revealing the activity state of the bioactive protein. Moreover, proteome-wide isolation of protein termini leads to generalized sample simplification, thereby enabling up to three orders of magnitude lower LODs compared to traditional shotgun proteomic approaches. We introduce the potential of protein termini for biomarker discovery, briefly review methods enabling large-scale studies of protein termini, and discuss how these may be integrated into a termini-oriented biomarker discovery pipeline from discovery to clinical application.