Proteolytic Signaling in Health and Disease

In recent years, powered by evolving technologies and experimental design, studies have better illuminated the regulating role of proteolytic enzymes across human development and pathologies. Proteolytic Signaling in Health and Disease provides an in-depth discussion of fundamental physiological and developmental processes regulated by proteases, from protein turnover and autophagy to antigen processing and presentation and major histocompatibility complex (MHC) molecules. Moving on from basic biology, international chapter authors examine a range of pathological conditions associated with proteolysis, including inflammation, wound healing, and cancer. Later chapters discuss the newly discovered network of connected events among proteases (and their inhibitors), the so-called ‘protease web’, and how best to study it. This book also empowers new research with up-to-date analytical methods and step-by-step protocols for studying proteolytic signaling events.

• Examines biological events triggered by proteolytic enzyme activity across human development and pathologies
• Discusses the role of proteolytic signaling in inflammation, wound healing, and cancer, among other disease types
• Features methods and protocols supporting further study of proteolytic signaling events
• Includes chapter contributions from international leaders in the field

• Language: English
• Publisher: Academic Press
• Release date: Oct 13, 2021
• ISBN: 9780323856973

Book preview

Preface

Proteases have leading roles in biology, and they caught my attention since the very beginning of my academic career, when I started studying the composition of snake venoms and the role of proteases in the pathophysiology of the envenomation. It turns out that, to my surprise, the main biological events observed after a hemorrhagic process (which are mainly caused by the metalloproteases present in snake venoms) are not only triggered by proteases but also sustained and amplified by such enzyme class. In fact, snake venom proteases were shaped by evolution, allowing the snakes to deal with their distinct biological constraints. Snake venom proteases were the starting point of my journey in the protease field. Since then, I have been studying the activity of proteases in different biological contexts and keep fascinated about the implications of their activities in biology.

Classification, evolution, and mechanistic studies on proteases are important sources of information for the understanding of their functions. However, a comprehensive understanding of the biological role of a given protease is only revealed by mapping the alterations triggered by them in signaling pathways. Yet this is quite challenging as there is a dynamic interplay of interactions in biological circuits; therefore, a substrate in one reaction may become an activated protease in the other (the activation of protease zymogens, in cascade-like pathways, clearly illustrates such a feature). Consequently, with everything in biology, understanding a given phenomenon often implies the understanding of various seemingly unrelated events.

From the very first glimpse of life, during egg fertilization, to cell death (in apoptosis), proteases are ubiquitous players in several metazoan signaling pathways. They all have in common the chemical reaction catalyzed (the hydrolysis of peptide bonds); however, the profusion of their biological effects is not easily anticipated and, more importantly, diverges strikingly even among protease isoforms and physiological states (i.e., health and disease states). Several factors contribute to this: structural diversity, which includes the existence of multidomain architecture in some enzymes (which is strongly correlated with their substrate diversity), the interaction with inhibitors, and more importantly, the fate of signaling pathways after the beginning of a proteolytic event.

Biological signaling processes have long been appreciated by investigators from all fields of biology, from homeostasis to pathological conditions. In this context, the study of proteolytic signaling in biological systems has been tremendously improved as high-throughput analytical approaches are emerging. Overall, Proteolytic signaling in health and disease intends to provide information on fundamental physiological processes regulated by proteases, from protein turnover and autophagy to antigen processing and presentation through MHC molecules. Pathological conditions will also be covered, including inflammation, wound healing, and cancer. Up-to-date analytical methods to study proteolytic signaling events will also be presented and discussed in this book. Collectively, the information contained in this book will make easier understanding of the set of interconnected events in which proteases (and their inhibitors) have leading roles, the so-called protease web, an important concept that wraps up the information contained in this book.

The interconnection nature of biological signaling events in which proteases have a role demonstrates that, indeed, the whole is something besides the parts. In other words, in essence, proteolytic signaling is the emergence of properties, from parts of a whole (interconnected) biological system and, in this context, cleaved substrates are as important as the proteases themselves for the proper biological signaling to occur.

Finally, I want to warmly acknowledge all the authors of this book for joining me in this endeavor and to have dedicated an important amount of their time to write their chapters, despite the worldwide pandemic SARS-COV-2 situation. Many thanks.

I hope you enjoy the ride on proteolytic signaling as much as we do!

Chapter 1: Proteolytic signaling: An introduction

Uilla Barcick; Maurício Frota Camacho; Murilo Salardani; André Zelanis    Functional Proteomics Laboratory, Department of Science and Technology, Federal University of São Paulo, São Paulo, Brazil

Abstract

Proteases account for almost 2% of every known genome and are ubiquitously required by different signaling pathways in living organisms. Unlike signaling pathways in which some reactions may be reverted by enzymes acting in opposing reactions, such as pathways regulated by kinases and phosphatases, the uniqueness of proteolytic signaling is mainly related to its irreversibility: proteases hydrolyze substrates, thereby resulting in the physical separation of portions in target proteins. Furthermore, limited proteolysis increases protein diversity both structurally and functionally, thereby also increasing the biological reachability of input signals in biological circuits. Hence, proteolytic signaling can be described as a biological signaling event that is not only triggered and regulated by proteolysis; it is a signaling pathway, in which the cleavage event may significantly affect the fate of target proteins and, more importantly, the biological outcome. Therefore, proteolytic signaling must be understood in light of the fate of processed substrates and their corresponding roles in biological circuits—the slight imbalance in protease web may answer for substantial deviations from health to disease states.

Keywords

Proteases; Proteolytic signaling; Degradome; Limited proteolysis; Systems biology

Acknowledgment

The work in the author's laboratory is supported by grants from Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP, grants # 2014/06579-3, 2019/10817-0, and 2019/07282-8).

Introduction

This chapter is intended to be a concise introduction to the subject of proteolytic signaling; the concept will be discussed considering some selected examples. There is no attempt to perform an exhaustive and descriptive explanation of signaling pathways in which proteases are central players; rather the focus is on the biological implications of such a particular signaling process. Specific biological processes that are somehow triggered or regulated by proteolysis will be presented and discussed in a comprehensive manner in the following chapters.

Proteases: A brief (and incomplete) introduction

Proteases are hydrolytic enzymes that are encoded by genomes from essentially every organism, from virus and bacteria to plants and vertebrates. Indeed, proteases account for almost 2% of every known genome [1]. Such enzymes are key effectors of several biological processes, ranging from the first glimpse of life in egg fertilization—a process in which acrosomal proteases in spermatozoon displays a role in breaking the egg’s glycoproteins matrix, in the zona pellucida—to cell death, in apoptosis (a regulated pathway mediated by a class of proteases termed caspases). Even though many biological processes rely on protease function, two important aspects related to protease activity need to be clarified beforehand: protein degradation and proteolytic processing are both performed by proteases, though they are not synonyms.

Protein content in any living organism is a result of the fine-tuned balance between two opposite rates: the rate of protein synthesis and the rate of protein degradation. In this respect, protein degradation is a broad term which, in eukaryotes, is mainly accomplished by the ubiquitin-proteasome pathway (discussed in detail in Chapter 2) and by lysosomal proteases, which are available in specialized cellular compartments, termed lysosomes. Therefore, under normal physiological conditions, these processes must be strictly coordinated to the cellular needs. On the contrary, proteolytic processing (or limited proteolysis) is a particular type of proteolysis where the cleavage of substrates is restricted to specific portions, frequently altering the activity of target proteins. Such a process is regarded as a posttranslational protein modification as it occurs in mature proteins (i.e., after their translation within living cells). Two main processes are involved in the modulation of protease activity: (i) the activation of protease precursors (zymogens) by limited proteolysis and (ii) their inactivation by the interaction with inhibitors. Hence, the interplay among proteases and their inhibitors, the protease web [2] (discussed in Chapter 11), has pivotal implications for both health and disease states in any living organism.

The chemical reactions by which a protein is broken down into peptide(s) vary among the distinct classes of proteases (i.e., serine, metallo, cysteine, aspartic, threonine, and glutamic proteases), and mechanistic details of such processes are beyond the scope of this book; however, a major determinant of any protease activity is its three-dimensional structure including, in multidomain proteases, the presence of ancillary domains (in addition to the protease domain), which are important for substrate targeting, kinetic properties, and cellular localization (Fig. 1A). In this context, the key point to understand protease roles in biological systems is to underscore its repertoire of substrates, the protease degradome (discussed in detail in Chapters 10 and 11), which may vary tremendously even among close-related protease families. Since the primary specificity of a protease is mainly determined by structural constraints related to the amino acids at the peptide bond that undergoes cleavage (the scissile bond), protease activity upon its substrates may be viewed as the protease’s footprint (Fig. 1B) and this feature is the fundamental basis for high-throughput analytical approaches to study protease cleavage sites in complex biological contexts (discussed in detail in Chapter 10).

Fig. 1Fig. 1

Fig. 1 Protease structure has main implications on its functions. (A) Structure of a hypothetical mature multidomain protease, evidencing its ancillary domains (A, B, and so on) which, in general, are involved in substrate targeting, kinetic properties, and cellular localization. Such additional domains are responsible for the substrate repertoire (degradome) of the protease. (B) Upper panel: the primary specificity of a protease ( gray ) is ruled by structural constraints at the scissile bond (displayed in red (dark gray in the print version), in the upper panel). Lower panel: the evaluation of the frequency of amino acids at P5 to P5′ in substrates (illustrated by the size of the letters in the y -axis) denotes the subsite specificity of a protease and might be regarded as the protease’s footprint and (B) presents the nomenclature by Schechter and Berger in which residues C-terminal to the cleavage site (scissile bond) are referred to as prime (P′), whereas N-terminal residues are referred to as non-prime (P) [3]. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Limited proteolysis and proteolytic signaling are seamlessly connected

Since some key points were introduced, we can now focus on the definition of proteolytic signaling, which can be described as a biological signaling event that is not only triggered and regulated by proteolysis; it is a signaling pathway, in which the cleavage event may significantly affect the fate of target proteins and, more importantly, the biological outcome. Unlike signaling pathways in which some reactions may be reverted by enzymes acting in opposing reactions, such as pathways regulated by kinases and phosphatases, the uniqueness of proteolytic signaling is mainly related to its irreversibility: proteases hydrolyze substrates, thereby resulting in the physical separation of portions in target proteins. Furthermore, cleaved substrates may affect downstream signaling pathways, acting in distinct ways according to the biological context (activation/inactivation of protein precursors or modulation of protein functions, for example). It is important to highlight, however, that proteolytic signaling pathways do not comprise proteolysis only; rather, proteolysis is an irreversible part of the process, although downstream reactions may eventually be performed by other proteins. For example, it has been shown that in some pathological conditions such as cancer, the cross talk between kinases and proteases is central for cancer progression [4]. The phosphorylation of proteases affects several important aspects of protease function, including their activation/inactivation, cellular location, changes in interaction partners, and their half-life. On the contrary, proteolytic processing of kinases also displays central regulatory roles, such as the removal of inhibitory domains, ectodomain shedding, and the generation of novel biologically active fragments [4].

There are several biological examples illustrating how limited proteolysis and proteolytic signaling are seamlessly connected; nevertheless, when considering these two interconnected events, there are three fundamental aspects that must be considered: [1] what substrate(s) is(are) cleaved [2], where it is cleaved and, finally, [4] the biological context underlying the signaling event. The following selected examples will be briefly discussed, along with their implications in biological systems.

N-terminal protein processing and proteolytic signaling

Proteases are ubiquitously required by several different signaling pathways in living organisms. For example, in both prokaryotes and eukaryotes, a number of intracellular proteins require the proteolytic processing of the initiator methionine for their maturation [5, 6]. Such a process is performed by methionine aminopeptidases (MetAPs) and often occurs while protein synthesis is still taking place, by the ribosomes in the cytosol; therefore, it is called a cotranslational modification process. In fact, the N-terminal region of MetAPs presents zinc finger-like domains that are important for proper ribosome association [7]. Although the proteolytic removal of initiator methionine in a large subset of nascent proteins is a process conserved from prokaryotes to eukaryotes, the biological roles of such aminopeptidases are beyond the maturation of nascent proteins. The inhibition of the human methionine aminopeptidase 1 activity in tumoral cells led to an accumulation of cells in the G2/M phase of cell cycle [8], suggesting a role for this aminopeptidase in the progression of the cell cycle. Interestingly, the proteolytic processing event catalyzed by MetAPs is significantly linked to other important signaling pathways related to protein turnover [9, 10]. In the middle of the 1980s, the group led by Prof. Alexander Varshavsky showed that the identity of the N-terminal residue of a protein is intrinsically related to its half-life [11], such a feature was named the N-end rule. In this respect, there are some N-terminal residues that may be regarded as degradation signals (N-degrons) that are, in turn, recognized by N-recognins, which are central components of the N-end rule degradation pathway. Consequently, amino acid residues at the N-terminus of a protein are referred to as stabilizing and destabilizing when proteins bearing such residues have long or short half-lives, respectively. In this context, it was observed that when amino acid residues at the second position that is normally not processed by MetAPs become exposed, they are recognized by an N-end rule-specific ligase and targeted to degradation via proteasome. On the contrary, residues that are often exposed by MetAPs are refractory to N-end-rule-mediated degradation. Therefore, the link between the MetAP specificity and protein turnover through the N-end rule has important implications for protein half-life, since the generation of stabilizing new N-terminus may prevent premature degradation by the N-end rule pathway [9]. Interestingly, in addition to protein turnover, cleavage of target proteins triggered by the N-degron-mediated degradation may also lead to important cellular events, such as inflammatory responses. A remarkable example is the downstream inflammation events triggered by the proteolytic activity of the enteroviral 3C cysteine protease, one of the main proteases encoded by human rhinoviruses. The 3C protease has an important role in cleaving protein precursors encoded by viral genome into individual components [12]. Interestingly, an expressive inflammatory response is initiated after the cleavage of the NACHT, LRR, and PYD domains-containing protein 1 (NLRP1) by the 3C protease. The protein NLRP1 acts as a sensor component of the inflammasome, a complex of multiprotein oligomers of the innate immune system, responsible for the activation of inflammatory responses. By cleaving NLRP1 at the Glu130-Gly 131 peptide bond, 3C protease exposes a neo-N-terminus which, in turn, becomes a glycine N-degron. The subsequent degradation of the processed NLRP1 by the proteasome leads to the generation of a C-terminal fragment that can activate the inflammasome, eventually contributing to the inflammatory disease of the airway caused by human rhinoviruses [13]. These brief examples illustrate how two seemingly unrelated biological events (protein maturation and protein turnover) are connected via proteolytic processing as well as some implications of the proteolytic events in each context.

Cleavage of protein precursors and proteolytic signaling

There are many situations where cleaved substrates may perform distinct biological functions depending on the cleavage site or on the biological context (i.e., a disease or a pathologic state). Furthermore, changes in the substrate structure after proteolytic cleavage may give rise to a plethora of effects in signaling pathways. Limited proteolysis increases protein diversity in terms of both structural features (due to structural alterations after proteolytic processing) and functional aspects (the new protein species can now execute distinct functions to respond to specific biological contexts). Indeed, this may be viewed as a parsimonious scenario for living organisms: a given biological function may be executed by a processed substrate (which was already available, although not functional) instead of synthesizing a new protein species to perform such a task. Therefore, in some cases, the proteolytically processed products may be functionally viewed as a completely distinct protein in comparison with its nonprocessed counterpart. Indeed, the alternative (new) functions of processed substrates increase the biological reachability of input signals in biological circuits (Fig. 2).

Fig. 2

Fig. 2 Biological reachability is increased by means of proteolytic processing of substrates in biological circuits. Proteases can modulate biological circuits through their direct activity upon substrates ( gray squares ) as well as indirectly ( colored squares ), when cleaved proteins eventually affect downstream reactions, resulting in distinct outcomes ( shaded rectangles ).

There are numerous examples where the imbalance in biological circuits due to proteolytic signaling results in striking outcomes. Perhaps one of the better examples of proteolytic signaling is observed is the classical sequence of proteolytic events (and their corresponding biological consequences) during the coagulation of blood. Such a pathway involves a series of subsequent proteolytic processing steps, performed mainly by sequentially activated zymogen form of serine proteases that ultimately lead to the cleavage of alpha and beta chains of the protein fibrinogen by the serine protease thrombin. As the coagulation process goes on, the successive cleavages of serine protease zymogens eventually amplify the initial signal by several orders of magnitude. In normal physiological conditions, the cleavage of Aα and Bβ fibrinogen chains by thrombin results in the generation of fibrinopeptides A and B, respectively, which in turn activates the transglutaminase (activated factor XIII, FXIIIa), which cross-links fibrin, thereby rendering the fibrin clot stable and insoluble. Interestingly, some thrombin-like enzymes isolated from snake venoms can directly cleave fibrinogen, however, usually releasing only fibrinopeptide A or B [14]. It turns out that, in the coagulation disorders observed after envenomation by some snakes, the resulting fibrin clot is not cross-linked by FXIIIa [14, 15]. Consequently, thrombin-like enzymes from snake venoms act as defibrinogenating agents; the activity of such serine proteases upon fibrinogen turns the blood nearly unclottable in vivo. This example illustrates the multifunctional roles of a single substrate after its cleavage by functionally related enzymes (i.e., thrombin and snake venom thrombin-like enzymes) in distinct pathophysiological conditions. Additionally, the venom of some snake species is rich in metalloproteases that are able to degrade extracellular matrix proteins, thereby promoting significant tissue damage in which hemorrhage is typically observed [16]. Synergistically, the proteolytic signaling triggered by snake venom proteases has important biological implications for prey subjugation, for example (further discussion on proteolytic signaling in snake envenomation will be presented in Chapter 12). In fact, these examples are just a snippet of a more complex biological scenario, which also demands the participation of several proteins (other than proteases).

Proteolytic signaling in health and disease

As a biological product derived from an intricate process of protein synthesis, protease expression is the result of the regulation of several pathways (transcription and translation, for example) acting coordinately and, more importantly, it is context-dependent. Furthermore, the main implications of protease activity in biological systems essentially rely on the fine-tuning mechanisms of protease inhibition, their biological availability (i.e., their expression status), and heritable diseases (mutations in protease genes, for example); proteolytic signaling, therefore, may result in distinct outcomes depending on the balance among all of these features which, in turn, mirrors the physiological state of the organism. As mentioned earlier, a number of ordinary physiological processes rely on proteolytic signaling. A classic example is the regulation of blood volume and systemic vascular resistance, which is performed by the renin-angiotensin system. By cleaving angiotensinogen at Leu43-Val44 peptide bond, the aspartic protease renin releases the decapeptide, angiotensin I. Further C-terminal removal of the two amino acid residues in angiotensin I by the zinc carboxy metalloprotease, angiotensin-converting enzyme (ACE) generates the octapeptide, angiotensin II, which promotes vasoconstriction [17]. In addition, ACE also degrades the potent vasodilator peptide, bradykinin, boosting the vasoconstrictor signaling initiated by renin [18]. Interestingly, an array of biologically active peptides generated from angiotensinogen have been described so far, all of them derived from the activity of proteases (i.e., renin, ACE, or ACE2) and with distinct functional implications in normal and disease conditions [19, 20].

Proteolytic signaling is also involved in the significant alterations in pathological conditions found in complex diseases such as cancer, for example. On this basis, proteases display a significant role in the process of metastasis, contributing to the effective spread of cancer cells to distant sites [21]. For example, increased activity of matrix metalloproteases (MMPs), namely MMP-1, MMP-2, and MMP-3, was reported in the set of secreted proteins derived from human gastric cancer-associated myofibroblasts [22], an important feature contributing to tissue remodeling of the cancer microenvironment. Qualitative and quantitative changes in the expression of proteases (mainly MMPs and ADAMs—A disintegrin and metalloproteases) are associated with the progression of melanoma, from nevi to radial and vertical growth [23]. Sandri and coworkers [24] showed that the resistance to the B-raf kinase inhibitor vemurafenib induced changes in the tumor microenvironment by the upregulation of MMP-2 and downregulation of its inhibitor, TIMP-2, in melanoma cells, with an increase in cell invasiveness. Laurent-Matha and coworkers [25] showed that proteolysis of the inhibitor of cysteine proteases, cystatin-C, by cathepsin D in the breast cancer environment enhanced extracellular proteolytic activity of cathepsins, contributing to cancer progression. Tumor growth and development depend on the cross talking between tumor and associated (stromal) cells. In this context, the interplay of surrounding nonmalignant stroma and/or infiltrating cells with tumoral cells has a pivotal role in the signaling events that take place within the tumor microenvironment. The activation of MMPs within the tumoral microenvironment, for example, is the result of, among other signaling events, proteolytic cascades generating autocrine feedback which includes the degradation of inhibitors, the activation of pro-MMPs, self-activation, and so on [26].

A close inspection on protease degradomes under distinct biological conditions (e.g., the profiling of proteolytic events in proteins secreted by a normal cell and its transformed phenotype) might shed light on the potential biological role of proteolytic signaling during oncogenesis [27]. Hence, the mapping and annotation of cleavage sites may reveal novel processed forms of substrates that, in turn, may be associated with the phenotypic plasticity found in the disease under investigation. Moreover, although the assignment of the protease(s) responsible for the proteolytic processing events in complex biological samples is challenging, the identification of proteolytic signaling exclusive to disease states are themselves signatures of a pathological state.

Overall, proteolytic signaling is more than meets the eye. More importantly, it is not only about protease activity; rather, it must be understood in light of the fate of processed substrates and their corresponding roles in biological circuits—the slight imbalance in protease web may answer for substantial deviations from health to disease states.

Outlook and perspectives

As an essentially irreversible biological event, proteolytic signaling represents an additional layer of complexity in the study of signaling pathways, which emerges not only from the dynamic interactions among proteins, or by the landscape of genetic mutations and/or altered protein expression, but also by means of the degradome status of a given set of proteins and under defined biological contexts. Since neo-N-termini are generated after proteolytic processing, such set of peptides/processed proteins may be targeted by analytical approaches, aiming at the prospection of markers in distinct physiological conditions (i.e., health and disease) and in several biological matrices such as tissue samples, plasma, saliva, and urine, for instance. The technology and analytical platforms to study proteolytic signaling events are evolving fast, allowing for the comprehensive profiling of protease degradomes in a timescale faster than ever seen before—this is critical for translational medicine, where personalized therapies are proving to be more effective than the traditional axiom: treating the disease, not the patient.

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Chapter 2: Ubiquitin ligases: Proteolytic signaling, protein turnover, and disease

Patrícia Maria Siqueira dos Passosa; Camila Rolemberg Santana Travaglini Berti de Correiaa; Caio Almeida Batista de Oliveiraa; Valentine Spagnol; Isabela Fernanda Morales Martins; Felipe Roberti Teixeira    Department of Genetics and Evolution, Federal University of Sao Carlos, Brazil

a These authors contributed equally for this work.

Abstract

The E3 ubiquitin ligases are the last step of the ubiquitination process in cells, being responsible for selection of the substrates. There are more than 600 E3 ligases in human genome highlighting their important role in different cellular processes. In addition to the E3 ligases, the ubiquitination process is catalyzed by the enzymes E1 (ubiquitin-activating enzyme) and E2 (ubiquitin-conjugating enzymes), and once polyubiquitinated, the substrate can follow three different ways: proteolytic degradation by proteasome, activation, or inhibition by ubiquitination without degradation, or it can also be deubiquitinated by deubiquitinating enzymes (DUBs). The kind of polyubiquitin chain introduced in the substrate will determine which way they will follow. Different cell signaling pathways or cellular processes are regulated by E3 ligases, such as NF-κB signaling pathway, mitophagy, interferon response, cell cycle, and DNA repair. And consequently, dysregulation of E3 ligases is related to diseases such as muscular atrophy, cancer, and neurodegenerative diseases (Parkinson’s disease, Alzheimer’s disease, and Huntington’s disease). Thus, the E3 ligases play an essential role in cellular