ABSTRACT

The Importance of B Research and Clinical Applications

David M. Kosa

Director: Amanda C. Sevcik, Ph.D.

Cathepsin B is a cysteine of the family. It plays an important role in intracellular , but it displays activity due to a unique structural element called the occluding loop. There is also a large amount of evidence that is involved in the development and progression of , Alzheimer’s disease, and pathological conditions. Throughout this thesis cathepsin B is analyzed for its role in pathology, mechanisms, and protease activity.

APPROVED BY DIRECTOR OF HONORS THESIS:

______

Dr. Amanda Sevcik

APPROVED BY THE HONORS PROGRAM:

______

Dr. Elizabeth Corey, Director

DATE: ______

THE IMPORTANCE OF CATHEPSIN B RESEARCH AND CLINICAL

APPLICATIONS

A Thesis Submitted to the Faculty of

Baylor University

In Partial Fulfillment of the Requirements for the

Honors Program

By

David M. Kosa

Waco, Texas

May 2020

TABLE OF CONTENTS

LIST OF FIGURES ...... iii

LIST OF TABLES ...... iv

DEDICATION ...... v

CHAPTER ONE – Foundation of Cathepsin B Research . . . . . 1

CHAPTER TWO – Enzymology of Cathepsin B . . . . . 5

CHAPTER THREE – Inhibitors . 17

CHAPTER FOUR – Proposed Mechanisms in Medical Research . . . 23

CHAPTER FIVE – Concluding Words ...... 28

BIBLIOGRAPHY ...... 29

ii

LIST OF FIGURES

FIGURE 1 ...... 5

FIGURE 2 ...... 7

FIGURE 3 ...... 9

FIGURE 4 ...... 10

FIGURE 5 ...... 11

FIGURE 6 ...... 13

FIGURE 7 ...... 15

FIGURE 8 ...... 17

FIGURE 9 ...... 22

FIGURE 10 ...... 24

FIGURE 11 ...... 25

iii

LIST OF TABLES

TABLE 1 ...... 8

iv

DEDICATION

To my loving family, my dear friends, my Thesis Director Dr. Sevcik, and the dedicated

professors of the Baylor science departments.

v

CHAPTER ONE

Foundation of Cathepsin B Research

Introduction to Cathepsin B

The lysosomal , cathepsin B, plays a valuable role in proteolysis, the breakdown of proteins into smaller components for recycling and cellular housekeeping. Cathepsin B “is of significant importance to cancer therapy as it is involved in various pathologies and oncogenic processes in humans.”1 However, the specificity of the compound, and the complex role of cathepsin B in cancer treatment has hampered the advancement of clinical application.2 Although researchers have spent decades studying cathepsin B, little is actually known about the mechanisms of it in regards to activation, regulation, and inhibition. Despite the lack of information, scientists have pursued research of cathepsin B in hopes of producing notable work in the field of cancer and disease treatment.

Historical Importance of Current Research

Cathepsin B has been the focus of numerous researchers and experiments since the late 1900’s. The first known documented report on cathepsin B is in 1957 by

Greenbaum and Fruton.3 Although they were only studying the purification and properties of the compound, their work began the cascade that would become crucial to the human understanding of . Shortly afterwards, Greenbaum joined with

Schoichet and Hirshkowitz to analyze the activation process of trypsinogen by cathepsin

B.4 A few other scientists joined in the initial research on the protease in hopes of

1 understanding the methods of activation; however, cathepsin B was mostly unknown during the 1950’s. The experiments in the early 1900’s were focused on the effects of as a whole with the occasional reference to a distinct “” in some articles with Fruton as a primary researcher. The 1952 article “On the proteolytic of animal tissues. Beef spleen cathepsin C” was the first article to mention a clearly distinguished compound known as cathepsin C.5 The second article on distinguished cathepsins was focused on the transamidation reactions that are catalyzed by cathepsin C.6 It was not until around the late 1970’s that cathepsin B became popularized across research groups and experiments. Part of this increase in popularity can be attributed to the Barrett assay of cathepsin B which was improved in 1976.7 The assay ultimately utilized the cleavage of the substrate benzyoyl-DL-arginine 2- napthylamide and the coupling of a diazonium salt with a napthylamine to generate a compound capable of being read at an absorbance of A520 nm.

With new techniques being developed, the number of articles being written on cathepsin B rapidly began to increase until researchers began to realize that the inhibition of cathepsin B could be crucial in preventing cancerous growth because of its role as an extracellular membrane degrader. The first inhibitor of cathepsin B, haptoglobin, was found by Snellmann and Sylvén; it is natural inhibitor of cathepsin B activity where haptoglobin is a protein that is responsible for binding to free hemoglobin in circulation.

However, it was not until the German scientists Appel, Huth, and Herrmann discovered the implications of cathepsin overexpression, that scientists began to realize that inhibition of cathepsin B could be a viable strategy for treating a variety of diseases and . Appel et al.’s experiment involved the serum testing of 43 children with varying

2 alterations of health. Of the children with inflammatory diseases, specifically leukemia and pneumonia, most of them experienced an increase in a variety of protein compounds including cathepsin B and cathepsin C.

Recent research with cathepsin B has provided a solid framework and guideline for future experimentation in the clinical field. Notably, the acceptance of cathepsin B being regarded as an effective biomarker for a plethora of cancers has pushed research advancement towards clinical experimentation.8–13 Research on cathepsin B involvement in cancer may be the most common, but there is almost an equivalent amount of evidence that it is involved in a wide variety of diseases and conditions. Given that cathepsin B is capable of crossing the blood-brain barrier and is involved in numerous pathological processes like , , and the production of toxic peptides, it is highly influential in neurological diseases.14 Specifically in reference to neurological diseases, cathepsin B can be tied to caused by apoptotic cell death, inhibitor treatments to prevent the decay of brain neurons and inflammation, and amyloid plaque production in

Alzheimer’s disease.15–17 With such an extensive repertoire of clinical implications, cathpesin B is definitely a highly valued research target and the focus of numerous experimentations.

Purpose of this Thesis

Overall, the majority of the early research on cathepsin B was severely limited by the lack of information on the structure, composition, and specificity of the cysteine protease. However, this limitation was overcome as technological advances and interest grew. Eventually, the mechanistic models of cathepsin B began to advance as more information on the protein became available. Although the mechanistic models have

3 improved, the current understanding of cathepsin B is still limited. Because cathepsin B is highly involved in the clinical research of cancer and neurodegenerative diseases, there are increasingly more research groups and individuals wanting to learn about cathepsin B and the best methods to manipulate the mechanism of action in favor of inhibition.

However, the complexity of cathepsin B has left significant gaps in the development of understanding cathepsin B. These gaps allow room for contradictory theories, and confusion in understanding the mechanisms of cathepsin B activity and regulation. The purpose of this thesis is to detail the comprehensive progress in the enzymology of cathepsin B while approaching theories and intricate mechanisms in order to show the potential of cathepsin B research and its clinical application.

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CHAPTER TWO

Enzymology of Cathepsin B

Structure

Because cathepsin B undergoes “considerable variation in its posttranslational modification,” it has been difficult to identify its numerous roles in “physiological and pathological processes.”18 The complete protein sequence was largely unknown until

1983 when Takio et al. published their work on rat liver enzymes.19 Today, it is known that the cathepsin B is located on 8, and it can be identified as the

CTSB Gene. The location of the gene can be found below in Figure 1.

Figure 1. Showing the Genomic Location of CTSB Gene in . The red line located between p23 and p22 shows the location of the CTSB Gene. The exact location is marked as 8p23.1 20

In 1991, Musil et al. were finally able to decipher the crystal structure of human liver cathepsin B through Patterson search and heavy atom replacement methods. The

Patterson function is equivalent to the Fourier transform of the intensities of the compound rather than the structure. The Patterson function was used in this approach because it is useful in solving the phase problem in X-ray crystallography. Shortly thereafter in 1996, Cygler et al. synthesized the inactive precursor of cathepsin B, procathepsin B. Both of these breakthroughs were pivotal in developing the fundamental

5 basis of cathepsin B specificity. Because of the X-ray data, Musil et al. were able to reconfirm the disulfide connectivities of bovine cathepsin B that were proven chemically correct by Bettie Evans and Elliott Shaw.21

Overall, Cathepsin B is a bilobal protein of approximately 30kDa with the and the substrate located between two large lobes of the protein.22 The precursor, procathepsin B consists of 43~46 kDa. When the mature cathepsin B is formed, the procathepsin B is split apart into two chains linked together by a disulfide bridge. The heavy chain of cathepsin B is approximately 25 to 26 kDa, and the light chain is approximately 5 kDa.20 The enzyme is synthesized as a preproenzyme of 339 amino acids, and it has a signal peptide consisting of 17 amino acids. When matured and modified, human liver cathepsin B is reduced to a sequence length of 205 amino acids on the heavy chain and 47 amino acids on the light chain.23 Musil et al. discovered the six disulfide bonds between positions 93-122, 105-150, 141-207, 142-146, 179-

211, and 187-198.23 Additionally, the final product undergoes another two amino acid modifications: a glycosylation at position 192 of the N-linked (GlcNAc) asparagine and the modified residue at position 220 of the N6-acetyllysine.24

6

Figure 2: Ribbon Plot of Cathepsin B Developed by Musil et al. This image is from Musil et al.’s experiment to map out the crystal structure of cathepsin B. The distinct crystal structure can be seen, showing many of the secondary and tertiary structures of the compound.23

The structure of human liver cathepsin B is well analyzed thanks to Musil et al.

Because of their work, the complete mapping of the modified product is easily available on Uniprot under the PDB ID 1HUC. The complete list of secondary structures are listed in the Uniprot table below in Table 1.

7

Table 1. Table of Cathepsin B Secondary Structures. A full list of the secondary structures present in the PDB ID 1HUC from human liver cathepsin B.20

Feature Amino Acid Position Beta strand 20-22 Helix 28-37 Beta strand 40-43 Helix 52-57 Beta strand 69-72 Beta strand 82-85 Helix 86-89 Helix 94-97 Beta strand 104-106 Helix 108-124 Turn 125-127 Beta strand 131-133 Helix 135-141 Helix 143-145 Helix 149-151 Helix 155-164 Beta strand 167-170 Turn 173-175 Beta strand 178-180 Beta strand 188-191 Beta strand 193-195 Helix 220-222 Beta strand 226-231 Helix 236-246 Beta strand 249-256 Helix 259-261 Beta strand 264-267 Beta strand 274-288 Beta strand 291-297 Beta strand 302-304 Beta strand 309-313 Turn 314-317 Helix 318-320 Turn 321-323 Beta strand 326-330

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Function

Cathepsin B is a lysosomal cysteine protease of the papain family where a is a “subcellular organelle that is found in nearly all types of eukaryotic cells.”25 Amongst the degradative enzymes are cysteine proteases which are enzymes that share a mechanism wherein a nucleophilic cysteine thiol is involved in a catalytic system.

Figure 3: The Lysosome of a Cell. The lysosome is the key organelle involved in the digestion of biomolecules. They contain hydrolytic enzymes which allow them to dispose of obsolete or damaged materials in the cell.25

Cathepsin B is also a known component of intracellular protein catabolism, and it is part of many processes such as: antigen creation during immune response, bone regeneration, and hormone signaling. 22 In antigen creation, “exogenous antigens are processed by lysosomal proteases” inside of antigen-presenting cells to make antigenic peptides.26 Cathepsin B is generally regarded as maintaining both and exopeptidase activity in protein turnover; however, recent studies have shown that it is

9 involved in the processing of amyloid precursor protein which has been linked to

Alzheimer’s disease.

Figure 4: Involvement of Lysosomal Cathepsins During and Apoptosis. This figure from Stoka et al. depicts the regulation that occurs from lysosomal cathepsins during the aging process and neurodegeneration.27

10

Because extracellular membrane degradation is a highly important factor in the development of many diseases, it is important to understand the entire functionality and cleavage of the structural and functional proteins affected by extracellular cathepsins. The proteolysis of ECM proteins occurs under biological conditions; however, most extracellular substrates of cysteine cathepsins are actually identified in in-vitro studies.

Despite the lack of in-vivo studies, researchers are confident in the extracellular involvement of cathepsins.

Figure 5: Extracellular Roles of Cysteine Cathepsins. Cysteine cathepsins are involved in numerous extracellular activities. Additionally, cathepsins are involved in a positive feedback loop wherein the production of cathepsins will lead to the cell signaling cycles in the microenvironment of different cells, including tumor cells that increase the production of cathepsins.28

Cysteine cathepsins were first linked to ECM proteolysis in cancer through cathepsin B in 1981.29 However, the exact roles and mechanisms of the specific cysteine cathepsins are still not completely understood today. The function of the endopeptidase and exopeptidase activity is explained more in the section Cleavage and the Catalytic

Triad.

11

Cleavage and the

When cathepsin B acts as an endopeptidase, it favors a “large hydrophobic side- chain in the substrate” composed of “two residues N-terminal to the scissile bond”.22

However, cathepsin B is considered a promiscuous enzyme compared to other lysosomal cysteine proteinases. In this regard cathepsin B is capable of accepting an arginine at this position because of the E245 glutamic acid located in the tip of the binding site. Because

E245 can act as an acceptor for the arginine side chain’s positive charge, the binding capability of cathepsin B is greatly expanded.

12

Figure 6: Mature Procathepsin B. The view of the molecular surface of the procathepsin B active site is shown in the front. The occluding loop can be seen just above the occluding-loop crevice. The structure of procathepsin B was provided by Cygler et al. 30

When cathepsin B acts as an exopeptidase, the structural importance of the enzyme’s occluding loop becomes apparent. As a peptidyldipeptidase, cathepsin B cleaves dipeptides at the C-terminus of peptides. The occluding loop, a unique structural element of cathepsin B, is responsible for the exopeptidase activity. The occluding loop is able to partially block the end of the active site and is responsible for positioning the

H111 positively charged imidazole group to accept the negatively charged carbon terminus of the substrate.

13

Although the occluding loop’s primary function belongs with the exopeptidase activity of cathepsin B, it is also believed that the occluding loop could potentially impact the endopeptidase activity. Notably, the endopeptidase activity of cathepsin B is less effective than some other enzymes in the papain family. The explanation for this deficit can be found in Cygler et al.’s analysis of the occluding loop.31 According to this proposal, the occluding loop is flexible and can adopt a conformation where it blocks the binding cleft. Thus, the reduction in effective endopeptidase activity is due to the “energy cost of altering the conformation of the occluding loop”.

Cathepsin B contains a catalytic site of three amino acids, cysteine, histidine, and aspartic acid, between the two lobes of the protein. 23 This site is commonly referred to as a catalytic triad. A catalytic triad consists of three coordinated amino acids wherein there is typically a motif of an acid, base and a nucleophile across the amino acids.32 In the case of Cys-His-Asp catalytic triads, the aspartate tends to have a significantly decreased effect because of cysteine’s low pKa. Thus, in the papain family of protease triads, the function is primarily encompassed in a Cys-His dyad. Additionally, in papain protease triads, the cysteine is deprotonated before catalysis occurs. The catalytic triad is most commonly used to cleave peptide bonds, asparagine, esters, and β-lactams; however, the activity of cathepsin B is a .28 In the protease enzyme carboxypeptidase, a protein or peptide is cleaved at the carboxy-terminal. This is in contrast to where cleavage occurs at the N-terminus. The generic mechanism of the catalytic triad can be seen in Figure 7.33

14

Figure 7: Catalytic Triad Mechanism of Proteolysis. Proteolysis begins with nucleophilic substitution at the carbonyl by the nucleophile. Then the acyl-enzyme intermediate is attacked by the second substrate, generating the second tetrahedral intermediate. Redistribution of electron charge kicks off the nucleophile amino acid group and forms the product.

In cathepsin B mediated bond cleavage, the reaction is catalyzed by the C29 cysteine residue on the left lobe when it interacts with the H199 histidine residue on the right lobe of the enzyme.23 In this environment the C29 thiol and H199 imidazole side- chains form an ion pair. Then, cleavage of the peptide bond is mediated by the nucleophilic attack of the negatively charged sulfur on C29 onto the carbonyl carbon atom.22 The process then goes through a clean-up stage where proton donation from

H199 onto the sulfur releases the enzymes from the final products.

Assays

This portion of the chapter covers the assays that are pertinent and most useful for cathepsin B research. As previously mentioned, the growth of cathepsin B research in the late 1900’s was primarily due to the easier procedures and assays being developed.

Notably, the Barrett assay and its updates allowed for an easy fluorometric assay of cathepsin B through the substrate benzyloxycarbonyl-phenylalanyl-arginine-4-methyl-7- coumarylamide.34 Currently, there are easier procedures to determine the enzymatic activity of all products with a specification for cathepsin B activity.35–37 Additionally, these advances in procedures also apply to inhibitor screening assays.38,39 Throughout the

15 most prevalent inhibitor screening assays, there is the common usage of the same inhibitor, E-64.

16

CHAPTER THREE Inhibitors

Figure 8: Types of Reversible Inhibition. The image depicts the possible sequenced pathways for the four differing types of reversible inhibition.40

17

Types of Inhibitors

Because high concentrations of cathepsin B can be indicative of disease or the promotion of disease and cancer development, a large portion of cathepsin B research is focused on finding inhibitors of the enzyme. In general, enzyme inhibitors are substances which can alter the catalytic process of the enzyme by either slowing down the process or completely stopping catalysis. There are four kinds of reversible enzyme inhibitors: competitive inhibition, uncompetitive inhibition, non-competitive inhibition, and mixed inhibition.

Competitive inhibitions is the process wherein a similar compound to one of the normal substrates of the enzyme binds to the active site of the enzyme, preventing the enzyme from binding to the normal substrate. Thus, an inhibitor and a substrate cannot bind to the enzyme at the same time. In competitive inhibition, an increase in inhibitor will show an increase in the effective competition for the enzyme’s active site. In kinetic analysis this inhibition will present no change to the maximum velocity of the catalytic rate and the appearance of an increased KM due to the lack of enzyme affinity for substrates when bound to the inhibitor.

Non-competitive inhibition is the process wherein an inhibitor binds to a separate site on the enzyme, causing a reduction to the affinity of the enzyme. Because non- competitive inhibition is not directly affected by the concentration of the substrate, it is extremely useful in experimental settings with varied substrate concentrations.

Kinetically, this means that the maximum velocity of the catalytic rate will decrease due to the enzymes lack of ability for the reaction to proceed efficiently. However, KM will

18 stay at the same value because the binding of the substrate to the enzyme remains functional.

Uncompetitive inhibition is the process wherein the substrate-enzyme complex becomes bound by the inhibitor. This will cause the maximum velocity to decrease as a result of the removal of the activated complex. Meanwhile, the KM will decrease due to the improved binding efficiency and removal of the ES complex. Thus, the kinetic value indicates a higher bonding affinity.

Mixed inhibition is the process wherein both the inhibitor and the substrate could bind to the enzyme, but the binding of either substance affects the affinity for the binding of the remaining component. Because the binding of either compound can happen first, some of this inhibitory effect could be reduced with increasing substrate concentration, but it will not eliminate all of the inhibition. Mixed inhibition typically occurs when an inhibitor binds to an allosteric site on the enzyme that is not the active site, causing a conformational change of the enzyme. This conformational change is capable of altering the affinity for the EI complex to the substrate, effectively reducing the amount of substrate bound.

Irreversible inhibition, also known as covalent inactivation, is a process wherein the inhibitor usually covalently modifies an enzyme causing the inhibition to not be reversible. Most irreversible inhibitors utilize a reactive functional group in order to form covalent adducts with the side chains of amino acids.41

19

Inhibitors of Cathepsin B

Inhibitors of cathepsin B are a highly valued field of research given that cathepsin

B is an abundantly expressed cysteine protease involved in many important pathological conditions and processes, such as cancer, Alzheimer’s disease, inflammation, and disease progression. Although many potential inhibitors have been tested under both in vitro and in vivo conditions, there is a limited number of reliable inhibitors of cathepsin B depending on the criteria of the clinical or experimental conditions. For example, one of the first discovered naturally produced inhibitors of cathepsin B and similar proteases was C. The two-step mechanism of inhibition of cathepsin B by cystatin C occurs through the initial binding of the N-terminus of cystatin C to cathepsin B followed by a large conformational change caused by the displacement of the occluding loop.42

However, the occluding loop is also the property of cathepsin B that allows it to perform exopeptidase activity. Although it is possible for to cystatin C to bind and inhibit cathepsin B, the conformational change of the occluding loop exerts stress on the inhibition mechanism. Thus, the characteristic of cathepsin B being less susceptible than other cysteine proteinases is due to the occluding loop. Overall, this complication in the inhibition mechanism reduced cystatin C’s effective inhibition on cathepsin B, meaning that other effective inhibitors were sought in favor over cystatin C. Other acceptable inhibitors of cathepsin B are antipain dihydrochloride, CA-074, inhibitor I, calpain inhibitor II, chymostatin, E-64, leupeptin trifluoroacetate salt, procathepsin B fragment 26-50, and Z-Leu-Leu-Leu-fluoromethyl ketone.43 Notably, the E-64 protease inhibitor has proven to be a valuable inhibitor of cathepsin B. The E-64 inhibitor is an irreversible and highly selective cysteine protease inhibitor of potent activity. The E-64

20 inhibitor is extremely useful for in-vivo studies as it maintains specific inhibition without inhibiting serine proteases like most cysteine protease inhibitors. Additionally, it has low toxicity and is permeable in cells. Due to its effectiveness E-64 protease inhibitor is the most commonly used inhibitor in cathepsin B enzymatic activity assays.

New Options

Recently, researchers Zhang et al. attempted to direct the inhibitor research of cathepsin B towards the development of a “humanized antibody inhibitor of cathepsin

B”.44 In an effort to reduce the lack of pharmacological activity and specificity of most cathepsin B inhibitors, they designed and developed a human antibody inhibitor that targets the CTSB gene. The antibody was developed through the fusion of procathepsin B and CDR3H, the complementary determining region of the Herceptin heavy chain.

Herceptin is also known by the generic name of Trastuzumab. It is a monoclonal antibody currently being used to treat metastatic breast cancer. Specifically, the antibody shows effective activity by targeting the HER2/neu protein receptor on tumorous cells.

The generation of the anti-CTSB antibody inhibitor produced similar results to the clinical reports of the parental Herceptin antibody. The newly formed antibody has high specificity at the nanomolar level for inhibiting the proteolytic activity of cathepsin B.

Additionally, the researchers were able to perform pharmacokinetic studies on mice and determined that the plasma half-life of the antibody cathepsin B inhibitor was approximately 42 hours. Overall, this discovery shows that efficient generation of human antibody inhibitors is possible for cathepsin B. Thus, future studies could utilize these resources to test a specialized human antibody for cathepsin B against diseases thought to be caused by overexpression of cathepsin proteases.

21

Figure 9: Anti-CTSB Antibody Inhibitor. A brief diagram for the spatial representation of procathepsin B being modified onto the Herceptin targeting antibody.40

22

CHAPTER FOUR

Proposed Mechanisms in Medical Research

One way to mechanistically study cathepsin B is to analyze the progression and development of diseases correlated to the presence of cathepsin B. Angiogenesis, cancer, osteoarthritis, rheumatoid arthritis, osteoporosis, and Alzheimer’s disease are known to have cathepsin B or their extracellular substrates involved in the progression of the disease. Although precise mechanisms are not known for every condition and disease linked to cathepsin B, further research into these mechanisms will allow cathepsin B research to gain more understanding on the intricate details of cysteine protease mechanisms and more importantly the broad specificity and conformational changes caused by the occluding loop.

Angiogenesis

In angiogenesis the cleaved targets of cathepsin B are ELR chemokines

(glutamate-leucin-arginin motif), non-ELR chemokines, and CD18 (integrin beta chain protein).28 Cathepsin B activates ELR chemokines and inactivates non-ELR chemokines where ELR-positive CXC chemokines induce migration of neutrophils and ELR-negative chemokines act as a chemical attractant for lymphocytes.45 Additionally, Nakao et al. found the cathepsin B mediation of CD18 to regulate the leukocyte recruitment from angiogenic vessels.46

23

Figure 10: Regenerative Angiogenesis.47 Angiogenesis is the process wherein new blood vessels are created from pre-existing blood vessels. Tumor growth and metastasis depend on angiogenesis and lymphangiogenesis triggered from tumor cells.48

Cancer

Closely correlated to the misregulation of angiogenesis is cancer and the development of tumor cells. The cleaved targets of cathepsins in cancer are Tenascin-C, nidogen-1, fibronectin, osteonectin, laminin, periostin, and collagen IV.28 Cathepsin B is not only critical to malignant progression, but it is also detectable as procathepsin B on the surface of tumor cells through an interaction with annexin II tetramer.49 Because of the interaction with the annexin II tetramer, other extracellular matrix proteins, like collagen, fibrin, and tenascin-C, are involved in the progression of gliomas.50

24

Osteoarthritis and Rheumatoid Arthritis

In osteoarthritis and rheumatoid arthritis the cleaved target of cathepsin B is aggrecan.28 Aggrecan is involved in the function of articular cartilage. Specifically, the proteoglycan provides a hydrated gel structure capable of distributing cartilage with weight-bearing attributes. The specific amino acid targets of cathepsin B on aggrecan are

Asn341-Phe342, and Gly344-Val345.51

Figure 11: Aggrecan and the Degradation Pathway. 52 An illustration of the principles of cartilage biomarkers.

Aggrecan monomers are first synthesized by chondrocytes. Then they aggregate onto hyaluronan before following a degradation pathway wherein cathepsin B is involved in the degradation and eventual release of the fragments into the synovial fluid. Once processed these fragments become crucial in the formation of cartilage.

25

Osteoporosis

In osteoporosis the cleaved targets of cathepsin B are osteonectin and osteocalcin.28 The specific amino acid targets of cathepsin B on osteonectin and osteocalcin are Arg44-Phe45.

Alzheimer’s Disease

Perhaps one of the most controversial topics is the role of cathepsin B in the progression and development of Alzheimer’s disease. With conflicting studies and experimental trials it is no wonder why the broad research on cathepsin B in the study of

Alzheimer’s disease remains inconclusive. Overall, there are both positive and negative attributes of cathepsin B involvement in Alzheimer’s disease pathology. Cathepsin B is positively involved in the lowering of Alzheimer’s disease pathology through an increase in amyloid degradation where the amyloid plaques are mostly believed to be either indicative or the cause of Alzheimer’s disease.53 The negative aspects of cathepsin B involvement in Alzheimer’s disease are twofold. The first problem is that cathepsin B may be contributing to the development of Alzheimer’s disease by acting as a beta- secretase. The traditional beta-secretase is BACE1, beta-site amyloid precursor protein cleaving enzyme 1. It functions to cleave the amyloid protein plaques at the WT beta- cleavage site. However, cathepsin B was shown to cleave the site about 400 times more effectively than the normal beta-secretase, creating more alpha beta proteins which are believed to be responsible for some of the symptoms of Alzheimers disease like memory deficiency.54 Additionally, in vivo studies of guinea pigs by Hook et al. showed that inhibition of cathepsin B strongly reduced amyloid levels and memory deficiency.17,55

However, cathepsin B is involved in a more thoroughly tested and negative aspect of

26

Alzheimer’s disease pathology through its ability to generate pyroglutamate Aβ. These peptides are N-terminally truncated alpha beta peptides in which the nitrogen terminus becomes cyclized. These peptides located in amyloid plaques are more “stable, neurotoxic, and [cause] more aggregation of Aβ than does full length Aβ” peptides.56

Additionally, Hook et al. also revealed in their research that “deletion or overexpression of the [cathepsin B] gene decreased or increased, respectively, the levels of the molecular species” pyroglutamate Aβ peptides, full length Aβ peptides, and pyroglutamate Aβ plaque load.17 Overall, cathepsin B is involved in negative aspects of Alzheimer’s disease pathology that are more dangerous and required intervention. Additionally, the positive aspect of cathepsin B is only useful at preventing lysosome accumulation, meaning that the intervention has to occur at the onset of suspicion. Thus, intervention would occur years before the actual manifestation of Alzheimer’s disease which is both impractical and unethical. Given these shortcomings and the benefits shown by Hook et al. in the inhibition of cathepsin B during the pathology of the disease, it is best to assume that synthetic inhibition of cathepsin B should occur during Alzheimer’s disease pathology.

27

CHAPTER FIVE

Concluding Words

As a member of the papain-like family, cathepsin B contributes many similar undesired consequences of cysteine proteases. Among these problems is the overexpression of endopeptidase activity; however, cathepsin B is also capable of exopeptidase activity due to the occluding loop, a unique structural feature. In endopeptidase activity, cathepsin B favors large hydrophobic side chains, but it is capable of accepting arginine in this same site furthering its broad activity. Cathepsin B has cellular functions in, antigen processing, apoptosis, inflammation, and pro-enzyme activation, but it has also been linked to numerous pathologies of diseases and malicious conditions. Overexpression of cathepsin B in cancers, Alzheimer’s disease, arthritis, and cardiovascular diseases all show increases in the development of the pathology, but inhibition of the enzyme has also proven useful to reduce both symptoms and the progression of the diseases. Overall, cathepsin B has been difficult to identify because of the numerous posttranslational modifications and physiological and pathological processes that are employed by the CTSB gene. Although the overexpression of cathepsin B continues to cause problems in numerous pathologies, there is promising evidence for the creation of specialized inhibitors capable of in vivo clinical trials.

28

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