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List of Papers
This thesis is based on the following papers, which are referred to in the text
by their Roman numerals.
I. Eriksson, S.; Gutiérrez, O.A.; Bjerling, P.; Tomkinson, B. (2009) Development, evaluation and application of tripeptidyl-peptidase II sequence signatures. Archives of Biochemistry and Biophysics,
484(1):39-45
II. Lindås, A-C.; Eriksson, S.; Josza, E.; Tomkinson, B. (2008) Investigation of a role for Glu-331 and Glu-305 in substrate binding of tripeptidyl-peptidase II. Biochimica et Biophysica Acta, 1784(12):1899-1907
III. Eklund, S.; Lindås, A-C.; Hamnevik, E.; Widersten, M.; Tomkinson,
B. Inter-species variation in the pH dependence of tripeptidylpeptidase II. Manuscript
IV. Eklund, S.; Kalbacher, H.; Tomkinson, B. Characterization of the
endopeptidase activity of tripeptidyl-peptidase II. Manuscript
Paper I and II were published under maiden name (Eriksson). Reprints were
made with permission from the respective publishers.
Contents
Introduction ..................................................................................................... 9
Enzymes ..................................................................................................... 9
Enzymes and pH dependence .............................................................. 11
Peptidases ................................................................................................. 12
Serine peptidases ................................................................................. 14
Intracellular protein degradation .............................................................. 15
The ubiquitin-proteasome system ........................................................ 15
Cytosolic peptide degradation ............................................................. 17
MHC class I antigen presentation ........................................................ 18
What is TPP II? ........................................................................................ 18
TPP II forms a gigantic complex ......................................................... 18
TPP II has a widespread distribution ................................................... 19
Why work with TPP II? ........................................................................... 20
TPP II is implicated in many cellular processes .................................. 20
TPP II has different substrate specificities .......................................... 21
TPP II is a potential drug target ........................................................... 22
Present investigation ..................................................................................... 23
What defines the primary structure of TPP II? (Paper I).......................... 23
Why does TPP II release tripeptides? (Paper II) ...................................... 25
Purification of TPP II (Paper II and III) ................................................... 27
How does the kinetic behaviour of TPP II vary with pH? (Paper III) ...... 29
What is the substrate specificity of the endopeptidase activity of TPP II?
(Paper IV) ................................................................................................. 30
Conclusions ................................................................................................... 32
Future perspectives ....................................................................................... 33
Sammanfattning på svenska .......................................................................... 34
Acknowledgements ....................................................................................... 37
References ..................................................................................................... 39
Abbreviations
3T3-L1 Fibroblast cell line with capability to differentiate into adipocytes
AAA-pNA Alanyl-alanyl-alanyl-paranitroanilide
AAF-pNA Alanyl-alanyl-phenylalanyl-paranitroanilide
BRCT Breast cancer type 1 susceptibility protein C-terminal
CD8+ cytotoxic T-lymphocytes
EL4 Mouse lymphoma cell line
ER Endoplasmatic reticulum
HEK293 Human embryonal kidney cells
IMAC Immobilized metal ion affinity chromatography
MHC Major histocompatibility complex
Tpp2 the gene encoding tripeptidyl-peptidase II
TPP II Tripeptidyl-peptidase II, species is indicated with lower-case letter,
i.e. mTPP II, murine TPP II, hTPP II, human TPP II, dTPP II, TPP II from
Drosophila melanogaster
Introduction
Proteins are some of the most diverse components of life. Consider the estimated number of species on earth, five million. In every genome, there are
perhaps ten thousand genes encoding at least one protein. Splicing variants
and posttranslational modifications add to the variety, leaving the total number of unique proteins somewhere in the vicinity of 1011. This might give the
impression that we all consist of a chaotic soup of proteins. On the contrary,
there is a high degree of organization in living organisms. The rate of production of each protein is fine-tuned from the gene level, where transcription
is tightly controlled, to the likewise precise mRNA maturation and translation levels. Once formed the proteins are affected by a plethora of factors
such as covalent modifications, cofactors and effectors, which can all determine the function of proteins and the activity of enzymes. The delicate balance between synthesis and degradation determines the concentration of
each protein, in turn deciding the fate of the cell. Ultimately, proteins are
degraded by a category of enzymes known as peptidases, an event that also
can be highly selective. This thesis concerns one such peptidase, tripeptidylpeptidase II (TPP II), and how structure relates to function in this enzyme.
Enzymes
Enzymes are proteins that catalyse chemical reactions, i.e. increase the rates
of transformation of one molecule into another, thereby directing the flow of
metabolites in cells. Catalysis decreases the activation energy, Ea, of the
reaction as depicted in Fig. 1. This can be achieved through stabilization of
the transition state, as is apparent for triose phosphate isomerase (1), or by
offering a different reaction route, e.g. as for pyridoxal phosphate dependent
transamidases (2).
9
Ea
Substrate
Product
Figure 1. Energy diagram for a simple reaction, the same reaction with lowered
activation energy (Ea), and divided in two steps.
For bimolecular or higher order reactions, i.e. reactions involving two or
more reactants, one of the main contributions of the enzyme might be binding the reactants in a favourable manner. This increases the local concentration of reactants and is sometimes referred to as the proximity effect (3). The
standard amino acid residues allow acid-base, covalent, nucleophilic and
electrophilic catalysis to occur, although sometimes non-standard amino
acids are needed, e.g. for glutathione peroxidase, where a selenocysteine
plays an important role in catalysis (4). For catalysis of redox reactions, cofactors such as heme groups and NADH are often utilized (5, 6).
k1
E+S
k-1
ES
k2
E+P
Scheme 1
Enzymology, the study of enzyme catalysis, started in the late 19th century, when scientist began to analyse the rate of alcohol fermentation in yeast
(7). It was established in the early 20th century that enzymes had hyperbolic
substrate dependence, as illustrated in Fig. 2. For the simplest enzyme catalysed reaction, i.e. Scheme 1, where the enzyme (E) binds substrate (S) and
releases the product (P), this behaviour can be described by the MichaelisMenten equation, developed by Leonor Michaelis and Maud Menten (8), [1]:
,
10
[1]
0
5
10
v0
20
30
where KM, the Michaelis constant, is (k-1+k2)/k1. In a reaction where k2 is
much smaller than k-1, KM represents the dissociation constant, KD, of the
enzyme/substrate complex. For more complicated reactions, the Michaelis
constant becomes correspondingly more complex, e.g. for epoxide hydrolase
(9). Despite this, KM is usually taken to correspond to the affinity of the
enzyme for the substrate, with a lower KM signifying a higher affinity. The
rate at which an enzyme saturated with substrate can catalyse a reaction
equals kcat, also named turnover number, which is the number of catalytic
events (or turnovers) per unit time. In the example above, where k2 <<k-1,
kcat is very close to k2 although, as for KM, increasing complexities in reactions pathways results in increased complexities in the expression for kcat.
Since enzymes are seldom saturated with substrate in a cellular environment,
kcat is rarely reached and might thus be viewed as a rather poor measure of
the real efficacy of the enzyme. Instead, the catalytic efficiency kcat/KM is
often used, as it describes how fast the rate of the catalysed reaction increases with increased substrate concentration at substrate concentrations
below KM.
0
200 400 600 800
[Substrate]
Figure 2. The dependence of the initial rate of enzyme catalyzed reactions on substrate concentration is usually hyperbolic. The figure is based on data from Paper III.
Enzymes and pH dependence
Physiological conditions are usually assumed to have a pH around 7.5, and it
is hardly surprising to find that most cytosolic enzymes have their highest
activity close to this pH. However, in certain situations it can be more favourable for the cell if enzymes have lower pH optima, e.g. the degradation
enzymes of the acidic lysosome, which would wreak havoc in the cell if they
were released into the cytosol. Many lysosomal enzymes have a pH opti11
mum around 5, which corresponds to the pH in the lysosome, but renders
them far from efficient in the more neutral cytosol.
Furthermore, pH dependency studies have been utilized in enzymology to
characterize reactive groups. It has also been used to probe the microenvironment in the active site, investigating effects from distant residues (10). In
this way, scientists have been able to probe the activities of enzymes with
macroscopic detection techniques such as spectroscopy and fluorescence. In
this work, pH dependency studies were conducted to investigate variations in
substrate binding and rate of catalysis between enzymes from different species as well as between different substrates.
Peptidases
Peptidases are enzymes that catalyze the degradation of proteins by hydrolyzing peptide bonds. At neutral pH peptide bonds are kinetically very stable, with a t½ of at least 2 years (11). The reason for this stability is in part
explained by the double bond nature of the peptide bond, where the electrons
of the carbonyl bond are partially translocated to the amide bond by the electronegative nitrogen. This results in a sterical hindrance, as the planar amide
bond is less accessible to water. The hydrolysis of peptide bonds proceeds
through a tetrahedral intermediate in all peptidases studied (Fig. 3), but the
nucleophile conducting the initial attack on the carbonyl carbon varies.
Oxyanion hole
+
Tetrahedral intermediate
Acyl enzyme
Figure 3. Reaction scheme for hydrolysis of a peptide bond by a serine peptidase,
showing the tetrahedral intermediate, the oxyanion hole and the formation and hydrolysis of the acyl enzyme.
12
The nomenclature of peptide binding and hydrolysis is illustrated in Fig. 4.
The cleaved peptide bond is called the scissile bond, and the amino acid
sidechains toward the N-terminus are called P1, P2, P3 etc., while the
sidechains towards the C-terminus are named P1’, P2’, P3’ etc. The corresponding binding pockets in the enzyme are called S1, S2, S3 and S1’, S2’ and
S3’, respectively (12).
S3
S1
S 2’
P3
P1
P2’
+
-
P2
P1’
P3’
S 1‘
S 3’
S2
Figure 4. The nomenclature of substrate binding by peptidases. The scissile bond is
indicated by an arrow, and the residues and binding pockets are marked according to
(12).
Peptidases are classified according to the nucleophile making the initial attack, or the residue or atom involved in activating water to act as a nucleophile, into four major groups. The most common type of peptidases are serine peptidases, which utilize a serine residue as the nucleophile, resulting in
an acyl enzyme (Fig. 3). The serine residue is usually part of a catalytic triad,
as discussed below (13). Serine peptidases have many important functions in
the body, such as in the blood clotting cascade and in the digestive tract (14,
15). Like serine peptidases, cysteine peptidases also form a covalent bond
with the substrate as part of the reaction mechanism, this time involving a
cysteine residue. The active site of cysteine peptidases encompasses a cysteine and a histidine residue, which fill essentially the same role as the catalytic triad in serine peptidases (16). Caspases are examples of cysteine peptidases that are involved in regulation and execution of apoptosis (17)
Metallopeptidases rely on a metal ion tightly bound in the active site, often zinc, which acts as a Lewis acid and activates a water molecule, and
thereby can attack the scissile bond (18). Thus, no covalent intermediate
with the enzyme is formed. In humans, a large family of metallopeptidases
are responsible for degrading the extracellular matrix. The fourth group,
aspartate peptidases, are active at acidic pH, due to the two aspartate residues in the active site (19, 20). Many aspartic peptidases are lysosomal, although some also occur in the stomach fluids (21).
13
Other variants of peptidases exist, although they are rare. One exception
of note is the proteasome, which has an active site containing an N-terminal
threonine residue. This peptidase will be further discussed below.
In addition to this classification of peptidases by catalytic mechanism,
they are also categorised by substrate cleavage mode. Exopeptidases cleave
at a specific distance from either the free N- or C-terminus of the substrate,
usually one, but sometimes as much as nine amino acid residues from the
terminus (22). Aminopeptidases are exopeptidases dependent on a free Nterminus, while carboxypeptidases are dependent on a free C-terminus. Exopeptidases rely on an interaction with the terminus that contributes a large
portion of the energy for substrate binding (Paper II). In contrast, endopeptidases do not have a specified distance to either terminus, but generally rely
on binding energy from a longer stretch of substrate (12). It has been noted,
however, that exopeptidases may have an endopeptidase activity, although
with lower activity due to inefficient substrate binding (23-25, Paper IV).
Serine peptidases
The two main clans of serine peptidases are trypsin and subtilisin (26). These
have a similar catalytic triad, composed of an aspartate, histidine and serine,
positioned in the same orientation in the 3D structures (27, 28). However, the
catalytic residues do not occur in the same order in the peptide chain, and
there are few other similarities in the amino acid sequence or threedimensional structure. It has therefore been concluded that the two clans have
evolved independently through convergent evolution (29).
The function of the serine residue, as discussed above, is to perform the
nucleophilic attack on the carbonyl carbon of the substrate scissile bond. The
role of the histidine is to deprotonate the serine residue, and protonate the
amine leaving group (Fig. 3). The role of the aspartate has been disputed;
either it accepts a proton from the histidine via a low-barrier hydrogen bond
(30), or simply positions the histidine for interaction with the catalytic serine
and the tetrahedral intermediate (31, 32).
However, even though the catalytic triad increases reaction rates six orders of magnitude, the total rate enhancement is 3-4 orders of magnitude
greater (11, 33). The majority of this enhancement is thought to stem from
transition state stabilization by the oxyanion hole (Fig. 3).
Although there are other clans with a Ser-His-Asp-triad, such as the prolyl oligopeptidases (34), not all serine peptidases have this active site composition. For example, some viral proteases have a Ser-His-His triad (35),
and the sedolisins, or carboxy serine peptidases, utilize a Ser-Glu-Asp triad
(36). One example of the latter is tripeptidyl-peptidase I, a lysosomal peptidase with exo- and endopeptidase activity (25). Furthermore, a bacterial
family of peptidases related to -lactamases have a Ser-Lys dyad, and some
14
serine peptidases even have the N-terminus positioned so that it becomes
part of the active site (37).
Subtilisin-like serine peptidases
The clan of subtilisin-like serine peptidases, or subtilases, have been grouped
into six families (38). The subtilisin, thermitase, proteinase K and lantibiotic
peptidase families are only found in microorganisms, while the kexin family
serve as proprotein convertases in eukaryotes. The pyrolysin family is more
diverse, both in species occurrence and in sequence conservation. All members of the pyrolysin family have a C-terminal extension, but this extension
does not always show convincing sequence conservation, which might
question this grouping.
The majority of subtilases are excreted endopeptidases, and the most
thoroughly studied enzyme of this clan, subtilisin BPN’, has an extensive
substrate binding region with eight binding pockets (39). The oxyanion hole
is formed by a conserved asparagine residue and the peptide backbone of a
serine residue (40).
Intracellular protein degradation
Protein synthesis is one of the most energy-consuming pathways in anabolism, corresponding to 25-30% of the cellular oxygen consumption in mammals (41). Uncontrolled degradation is likely to be detrimental to the organism. Even so, up to 30% of the ribosomal products are defective and need to
be degraded (42). Cellular processes such as mitosis and apoptosis are
tightly regulated by expression of many proteins, which need to be degraded
at the correct instance. Hence, the degradation of the proteins inside a cell is
a very selective process. There are two major pathways for protein degradation in the cell, the lysosome and the ubiquitin-proteasome system. Proteins
are taken up by the lysosome either by macro- or microautophagy, where a
part of the cytosol is completely surrounded by membrane and devoured
(43). This self-eating process increases in starvation. Alternatively, proteins
might be specifically targeted for degradation in the lysosome by chaperonemediated autophagy (44). The ubiquitin-proteasome system targets proteins
for degradation in the cytosol or nucleus by polyubiquitylation , which results in peptides that are either processed to free amino acids, or presented
by MHC class I (45). Degradation of proteins by the ubiquitin-proteasome
system is much more selective, as described below.
The ubiquitin-proteasome system
Ubiquitin (Ub) is a small protein utilized in many processes in the cell, including signal transduction and endocytosis (46,47). The ubiquitylation is
15
initialized by the activation of Ub by E1, which forms a thioester bond with
the C-terminus of Ub (48). The thioester bond is transferred to the active-site
cysteine of an E2 protein, of which there are approximately forty in mammals. Selectivity is achieved when one of the thousand E3 proteins recognizes a misfolded, malfunctioning or superfluous protein and promotes the
attachment of the Ub molecule on a -NH2-group of a lysine residue in the
target (49). Elongation of the Ub-chain through E4 elongase leads to
polyubiquitylation, usually via the Lys-48 of the Ub moieties, which targets
the selected protein for degradation in the proteasome (50), (Fig. 5).
The 26S proteasome is a 2.5 MDa complex consisting of the 20S core
particle, which confers the proteolytic activity, and usually two 19S regulatory particles. Core particles consists of four rings with seven subunits each,
1-7 or 1-7 (51). The -subunits are positioned in the middle of the cylinder,
whereas the -subunits form the top and bottom, blocking entry into the interior of the core particle prior to association with the regulatory particle (52).
Regulatory particles consist of at least 19 subunits, 6 of which are ATPases,
whose combined function is to recruit, deubiquitylate and unfold polyubiquitylated proteins in an ATP-dependent manner (53). Unfolded proteins are
passed into the catalytic core particle, where degradation commences at the
active sites in the 1, 2 and 5 subunits (54, 55). Specificity differs between
the three active sites, conferring preference for aromatic, acidic or basic
amino acids in the P1 position (56). Once the peptide products are small
enough to exit, they diffuse out of the core particle, resulting in products 225 amino acids in length (57), (Fig. 5).
16
E2
E1
E3
E3
E2
Ub
Target
Ub
E1
Target
Ub
Ub
Target
Ub
Target
19S
E4
20S
Target
Ub
19S
Ub
TOP
LAP
BH
TPP II
Ub
Ub
Ub
Free amino acids
Figure 5. Protein degradation by the ubiquitin-proteasome system and cytosolic
peptidases. Ubiquitin is activated by E1 and transferred to an E2 protein. The protein
to be degraded (target) is recruited by an E3 protein, and this in turn promotes the
attachment of ubiquitin to the target protein. Following chain elongation by E4, the
target protein is recognized by the proteasome, deubiquitylated and degraded into
peptides. These are further degraded by peptidases such as thimet oligopeptidase
(TOP), tripeptidyl-peptidase II (TPP II), leucine aminopeptidase (LAP) and bleomycin hydrolase (BH).
Cytosolic peptide degradation
Proteasomal degradation products are further degraded by a diverse array of
peptidases in the cytosol (Fig. 5). Products of 9-17 amino acids can be degraded by endopeptidases such as the metallopeptidase thimet oligopeptidase
(TOP) into peptides of 6-9 amino acids (58). The cysteine peptidase bleomycin hydrolase (BH) can process peptides of up to 42 amino acids endopeptidolytically as well as by amino- and carboxypeptidase activities (59, 60).
TPP II, a cytosolic peptidase whose main activity is the removal of tripeptides from the N-terminus of peptides, has been shown to be required for
degradation of some peptides that are more than 15 amino acids (61). Other
aminopeptidases such as the metallopeptidases puromycin-sensitive aminopeptidase and leucine aminopeptidase (LAP) can conclude the process by
removing single amino acids (62, 63). The end products of cytosolic peptide
degradation are for the vast majority of peptides free amino acids that are
17
reused in protein synthesis, although some longer peptides are exported into
the ER lumen for presentation by the MHC class I, see below.
MHC class I antigen presentation
One of the major immunological defence systems in the body is the surveillance of cells by CD8+ cytotoxic T-lymphocytes (CTL). The CTLs recognize
antigens derived from intracellular proteins displayed on the cell surface by
major histocompatibility complex I. Peptides of 8-11 amino acids are either
imported directly from the cytosol, or generated in the ER by further trimming of products from the cytosolic protein degradation by the proteasome
or subsequent peptidases. These peptides are assembled onto MHC Class I
molecules by the multimeric peptide loading complex, and the MHC Class Iantigen complex is transported to the cell surface via the Golgi apparatus
(64). By this mechanism, the immune system can recognize cells containing
viral or microbial proteins, as well as cells displaying altered protein composition, such as in tumour malignancies (65).
What is TPP II?
When first reported, TPP II was found in rat liver, but has since been established as a more or less ubiquitously expressed cytosolic peptidase (66-68,
Paper I). As the name indicate, the main activity results in the release of
tripeptides from the free N-terminus of longer peptides, i.e. an aminopeptidase activity. In addition to this activity, an endopeptidase activity has also
been reported (69). Although this activity is three orders of magnitude lower
than the exopeptidase activity, for some substrates it is as efficient as the
proteasome. The catalytic domain of TPP II is subtilisin-like, with an almost
200 amino acid residue insert between the catalytic aspartate and histidine
(66). In addition, following the subtilase domain is an approximately 700residue extension, leaving the total peptide chain length at 1249 amino acids
in mammals.
TPP II forms a gigantic complex
The TPP II monomer is difficult to study in isolation, as dimers are formed
very rapidly (70). Dimers stack into long strings, which twist around each
other in pairs to form a large complex with a total of 16-20 dimers (Fig. 6).
This complex has a molecular mass of 4-6 MDa, depending on species, and
is approximately 50 nm in length (70, 71). To put this into perspective, most
proteins weigh less than 100 kDa and measure no more than a few nm.
18
Figure 6.The EM-structure of the dTPP II complex (EMD 1732, (72)). The complex
consists of two strands of ten dimers each, joined together at the end dimers to form
a 6 MDa particle.
The crystal structure of the TPP II dimer from Drosophila melanogaster was
recently solved (72). Unfortunately, the enzyme is crystallized in an inactive
form, having a misplaced loop in the active site and a distorted catalytic triad
resulting from a partial unwinding of the helix containing the serine residue.
This might be the reason for the severely decreased activity, approximately
one tenth, in dimers when dissociated compared to when in the complex
(73). The reversible dissociation and association of the complex has been
proposed to be a regulation mechanism, albeit a rather slow regulation (71,
73). Regardless if complex formation can be controlled by the cell, the low
activity of the dimers probably serves to protect the cell from unhindered
proteolysis, as the active site is rather well exposed in the dimer crystals. In
the complex, however, the active site must be reached through a chamber
system, which would effectively hinder any native proteins from reaching
the active site (72).
TPP II has a widespread distribution
Since first discovered in rat liver, TPP II activity has been reported in various tissues in other mammals, chicken, fruit fly, plant and yeast (67, 74-76,
Paper I). In fact, TPP II seems to have a ubiquitous distribution in animals
and plants, which promotes the idea that TPP II has a housekeeping function
19
in the cell. Despite this, removal of the TPP II in Arabidopsis thaliana or
Schizosaccharomyces pombe did not result in any physiological abnormalities (74, Paper I). In contrast, removal of the TPP2 gene in mice has a more
drastic effect. One group reported a failure to generate TPP2-/- mice due to
embryonic lethality in day 9.5 (77). Another group successfully generated
TPP II deficient mice, although these mice had decreased life spans and several defects in the immune system (78). These findings are discussed in more
detail below.
Why work with TPP II?
There are many reasons for studying the action of a specific enzyme. The
knowledge might be used to explain the physiological role of the enzyme, or
to elucidate the mechanism of binding and catalysis displayed by that enzyme. This knowledge is essential for drug discovery efforts, since mechanistic data is utilized to a large extent in modern drug design (79). For TPP
II, all these apply; there are numerous reports on the involvement of TPP II
in different physiological processes, but the exact role of TPP II is not
known. Furthermore, the mechanism of substrate interaction is not fully understood, and TPP II could be a drug target for more than one disease.
TPP II is implicated in many cellular processes
The physiological function of TPP II has so far been proposed to be degradation of peptides released by the proteasome into tripeptides that can be efficiently degraded by aminopeptidases into free amino acids (66) (Fig. 5).
However, this housekeeping function does not seem to be consistent with the
involvement of TPP II in processes such as apoptosis and cancer, as described below. Since no specific substrate for TPP II has been identified, and
the structure excludes the possibility of degradation of intact proteins, the
exact role of TPP II in these processes remains to be determined. It is likely
that a decrease in TPP II levels will result in an increased concentration of
peptides in the cell, in conjunction with decreased rates of release of amino
acids. This might result in secondary effects, such as decreased rates of protein production or disruption of protein-protein interactions (80, 81).
TPP II in apoptosis
The first indication that TPP II was involved in apoptosis control was a report that an activity inhibited by AAF-CMK promoted apoptosis induced by
Shigella flexneri invasion, ATP and staurosporine (82). In contrast to this
find, overexpression of TPP II in Burkitt’s lymphoma cells was found to
impart protection from apoptosis (83). In combination with decreased proteasomal activity, increased TPP II activity developed during tumour growth
20
in lymphoma and melanoma cells, which was interpreted as an adaptation to
avoid apoptosis (84).
In addition to these findings, overexpression of TPP II in HEK293-cells
has been found to increase growth rate and result in chromosomal aberrations and centrosome abnormalities (85). Knockdown of TPP II by siRNA
led to decreased growth rates and polynucleated cells (85). Purportedly, TPP
II allows cells to avoid apoptosis despite activated mitotic check points (86).
It has been reported that TPP II translocates into the nucleus as part of the
irradiation response (87, 88), although this is disputed (89). However, TPP II
depletion in mice results in activation of apoptosis in T-cells, and premature
senescence in fibroblasts, in conjunction with upregulation of p53 (78). Although the precise mechanism behind this is unclear, it is apparent that TPP
II contributes to the survival of cells that would otherwise undergo apoptosis.
TPP II in MHC class I antigen processing
Proteasomal products are often trimmed before loading onto the MHC Class
I complex. The role of TPP II in this process has been investigated in several
studies. For example, the HIV Nef74-82 epitope has been reported to be generated by endopeptidolytic cleavage by TPP II (90). Other epitopes have also
been reported to be dependent on TPP II (91, 92). For several epitopes, however, no involvement of TPP II could be observed (93-96), and sometimes a
destructive role was noted (97). In general, TPP II seems to be important for
processing longer proteasomal degradation products (14-17 amino acids and
more), but not shorter ones (61, 98-100).
TPP II has different substrate specificities
It is not known which of the exo-and endopeptidase activities of TPP II that
is important for the physiological role of TPP II in different situations. The
exopeptidase activity is quite well-charactarized, whereas the endopeptidase
activity has been less studied. In the exopeptidase activity, the N-terminal
amino group is bound and a preference for aliphatic or aromatic amino acids
in the P1 position has been noted (75, Paper II). The endopeptidase activity,
although much slower, has been reported to favour basic amino acids in the
P1 position (69). This apparent discrepancy raises questions about the substrate binding ability of TPP II, and whether the binding sites differ between
the two activities. For example, the absence of a binding interaction with the
N-terminal amino group of the substrate might open a possible binding interaction in a different portion of the active site. Alternatively, the active site
for the endopeptidase activity might be located at a completely different part
of the enzyme. However, no homology with any known peptidases has been
found elsewhere, which renders this possibility unlikely. Nevertheless,
studying the binding interactions for both the exo-and endopeptidase activi21
ties can give valuable information regarding the architecture of the active
site, and thus be of aid in the development of specific inhibitors of TPP II.
TPP II is a potential drug target
It has been reported that a membrane-bound form of TPP II has the ability to
degrade the neuropeptide cholecystokinin-8 (101). Since this peptide aids in
transmission of satiety signals, it was hypothesised that inhibiting TPP II
would decrease hunger or overeating. Indeed, injection of the specific TPP
II inhibitor butabindide reduced food intake in mice (101). Since butabindide is unsuitable as a drug candidate for obesity because of its low stability
in blood plasma, attempts have been made at producing more promising
molecules, although none has reached clinical trials as of yet (102-104).
There have been several attempts at producing cells and organisms with
reduced concentrations of or completely depleted of TPP II (74, 77, 78, 98).
One group has reported homozygos Tpp2-/- mice that were embryonic lethal
(77). Heterozygotes, however, had a compromised adipogenesis, a phenomenon also observed in the worm Caernohabditis elegans (77). The decreased adipogenesis could not be linked to decreased food intake, and thus
not to decreased degradation of cholecystokinin-8. Furthermore, in 3T3-L1
cells treated with RNAi against TPP II, decreased adipogenesis was also
observed, and this could be reversed by expression of a part of the Cterminus, indicating that the function of TPP II in this context was independent of enzymatic activity (77). This curious report again links TPP II to obesity, although apparently by a completely different mechanism.
Since TPP II has been shown to have an anti-apoptotic role in many different cancer cell types, it could be considered a drug target for cancer therapy. One group has reported that Z-GLA, a tripeptide with a blocked Nterminus, is a highly potent TPP II inhibitor (87). In combination with irradiation treatments, this molecule inhibited tumour growth. However, other
groups have since failed to confirm the TPP II-inhibiting capabilities of ZGLA (78). This leaves the question whether TPP II would be a good drug
target for cancer therapy open for further investigation.
22
Present investigation
May you live in interesting times – T. Pratchett
The overall aim of the present investigation was to gain insight into the function of TPP II, both on an enzymatic and a physiological level. With a vision
to one day fully understand the cellular function of TPP II, and to provide
knowledge for potential future drug development, these are the questions
addressed in this thesis:
What defines the primary structure of TPP II?
Why does TPP II release tripeptides?
How does the kinetic behaviour of TPP II vary with pH?
What is the substrate specificity of the endopeptidase
activity of TPP II?
By answering these questions, I hope to make a contribution to the understanding of this intriguing enzyme. I have personally been fascinated by the
dual exo/endopeptidase activity of TPP II, and how the substrate specificity
seems to differ between these. It has been very interesting to first investigate
one, then the other, trying to piece together how the active site might interact
with the substrate in the different situations, and what importance this might
have in the cellular environment.
What defines the primary structure of TPP II? (Paper I)
Tripeptidyl-peptidase II is a very large enzyme, not only with respect to the
quaternary structure, but also the peptide chain itself is quite extensive, with
approximately 1250 amino acid residues in mammals, and even longer in
some species, such as insects (76). The catalytic subtilisin-like domain only
constitutes 300-400 amino acids, and the rest of the sequence has little or no
similarity to any known protein. It was previously known that the insert between the catalytic aspartate and histidine, henceforth referred to as the DHinsert, had an important role in complex formation (105). In addition, part of
the C-terminal extension (amino acids 520 and onward) had been reported to
affect such varying functions as fat metabolism and nuclear translocation
23
(77, 87). Hence, it appeared important to investigate sequence similarities, to
see if there were any highly conserved regions that might have functional
importance.
Eleven regions of high evolutionary conservation were identified in a set
of 16 sequences selected from 58 TPP II sequence homologues found during
data harvesting. From these regions, signatures were created that were able
to separate TPP II homologues from other subtilisin-like serine peptidases,
such as pyrolysin (Paper I). Pyrolysin also contains a DH-insert and a Cterminal extension but does not have any of the functional traits that characterize TPP II (38). The signatures were very selective, and all TPP II homologues detected so far matched at least two signatures. Thus, these signatures
can be used as a tool for identifying TPP II homologues, which might yield
more accurate annotations in the future. Seven of the signatures covered
regions of previously noted functional importance, such as the catalytic aspartate and histidine residues, as well as part of the BRCT domain reported
to be important for nuclear translocation (87). Since the remaining four also
contains amino acid residues that are conserved in TPP II homologues from
all eukaryotic species, it was suspected that they also have a vital role in this
enzyme (Paper I). Indeed, when the crystal structure of TPP II from Drosophila melanogaster was published (72), it could be noted that two of these
conserved regions of unknown function were positioned in the interface between the catalytic domain and a C-terminal domain. Two highly conserved
tryptophan residues from two different regions were in close proximity. Further investigations will be needed to elucidate whether these two residues, or
other highly conserved residues, play an as yet unknown role in the structure
or function of TPP II.
TPP II homologues from human, mouse, rat, fruit fly and arabidopsis all
have a very large molecular size and degrades peptides into tripeptides (71,
74, 76, 106). In addition, a peptidase sharing these characteristics was purified from the yeast Schizosacharomyces pombe, although this was not believed at the time to be a sequence homologue (107). However, we were able
to prove that a genetically engineered S. pombe without the gene encoding a
TPP II homologue did not contain the tripeptidyl-peptidase-like activity
found in the unmodified yeast (Paper I). This demonstrated that there are
functional TPP II sequence homologues in the three main kingdoms of eukaryotes, and thus we made the assumption that any sequence homologues
found within this domain also shared a common function.
During the search for TPP II sequence homologues, it was evident that all
sequenced genomes from animals and plants contained at least one copy of a
gene encoding TPP II. In fungi, only some genomes contained a homologue,
while most protozoan did (Paper I). In our database search, we also came
across an enzyme from the bacteria Blastopirellula marina annotated as a
pyrolysin, which had very high sequence similarity to TPP II. Upon a phy24
B
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logenetic analysis, this enzyme was grouped with TPP II homologues from
plants and so it was hypothesised that it was the result of a horizontal gene
transfer (Fig. 7). Furthermore, another bacterial protein, from Salinospora
tropica, annotated as a TPP II homologue was grouped together with pyrolysin. In both cases, the signatures developed in this work gave more accurate annotations. Further investigation revealed that the protein from
B.marina did not appear to have any tripeptidyl-peptidase activity, which
might be guessed from the lack of a conserved glutamate residue important
for exopeptidase substrate binding (Paper II). It appears that since the TPP
II-like activity did not confer any fitness advantage in these bacteria, it has
slowly lost that function.
H
sa
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en
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oga
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Eukaryotes = Green
Bacteria = Red
Archea = Blue
Figure 7. Neighbour joining was used to perform a phylogenetic analysis on TPP II
homologues as well as other subtilases (cf. Paper I). Eukaryotic proteins are marked
in green, bacterial in red and archeal in blue. For TPP II homologues, the name of
the species in which the gene was found is given whereas for other subtilases, the
name of the enzyme is given with the species in parenthesis.
Why does TPP II release tripeptides? (Paper II)
It was evident at an early stage that TPP II was dependent on a free Nterminus in the peptide substrate in order to bind and affect hydrolysis (68,
75). Thus, it was hypothesised that the positively charged N-terminal amino
group interacted with one or more negatively charged amino acid residues,
25
forming a molecular ruler for “measuring” the substrate. It was found from
sequence comparison that three acidic amino acid residues positioned in or
around the potential S3 pocket were conserved throughout the known TPP II
homologues at the time (Paper II). To see the possible spatial arrangement of
these residues, a homology model was built based on the closest structure
found at the time, subtilisin BPN’. Two of the residues, Glu-305 and Glu331, were found to be positioned favourable for interaction with substrate so
as to provide a molecular ruler for the enzyme (Fig. 8).
Glu-331
Glu-305
Ser-449
Asp-44
His-264
Figure 8. The active site of a homology model of mTPP II, with the catalytic triad
and Glu-305 and Glu-331 shown as ball and stick, and the backbones of the S1
pocket-forming residues magenta. This model was constructed based on the crystal
structure of dTPP II and subtilisin BPN’ as described in Paper III.
To test if these glutamates indeed interacted with the N-terminus of substrates, mutated variants of murine TPP II (mTPP II) with the respective
glutamate exchanged for glutamine or lysine were expressed in Pichia pastoris and purified by IMAC (Paper II). Only one mutant variant, E331Q, had
sufficiently high enzymatic activity to allow kinetic parameters to be determined. It was found that KM was increased at least 100-fold in this mutant
compared to the wild-type enzyme, suggesting a quite significant decrease in
binding affinity for the substrate used. Furthermore, the inhibition of the
E331Q mutant by an octapeptide inhibitor was essentially unaffected by the
removal of the N-terminal amino group of this peptide. In contrast, for the
wild-type mTPP II, this change caused a 1000-fold increase in IC50. The
specific TPP II inhibitor butabindide, which was developed to bind the S3
pocket of TPP II, also had a drastically increased IC50. For the Glu-305 mutants, however, little or no activity could be detected, which might indicate a
drastic change in the structure of the active site (Paper II). Indeed, at least for
the E305Q mutant, a decrease in size was observed, which could reflect difficulties in complex formation. However, since the dissociated enzyme has
26
one tenth of the activity of the complex (73), this could not be the sole cause
of the low activity in this mutant variant.
When the crystal structure for dTPP II was solved, the glutamates corresponding to Glu-305 and Glu-331, i.e. Glu-312 and Glu-343, were found in a
similar position (72). The glutamate corresponding to Glu-305 was found to
form a hydrogen bond with a tyrosine residue in the C-terminal part of the
enzyme, which might be the reason for the complete loss of activity when this
residue was mutated in mTPP II. Mutant variants of dTPP II with glutamine
instead of glutamate in these two positions could form the complex but were
shown to have severely decreased activity, at least in part dependent on an
increase in KM (72). This confirms our theory that a molecular ruler exists in
TPP II, consisting of at least one glutamate. Other peptidases have also been
reported to have a similar mechanism of substrate binding, such as prolyl
tripeptidylpeptidase, dipeptidyl peptidase IV and ERAP1 (22, 23, 108).
In conclusion, it is now evident that the substrate binding mechanism of
mTPP II involves an interaction with Glu-331. This interaction positions the
peptide substrate such that the third peptide bond is cleaved, and thus it constitutes a molecular ruler, ensuring that the product released is a tripeptide.
Purification of TPP II (Paper II and III)
In order to study the kinetics of the exo-and endopeptidase activities of
TPP II, a new expression and purification system needed to be developed.
The system used for the glutamate variants provided a source of TPP II
where mutant variants could be expressed and purified without contaminations from endogenous, wild-type enzyme, which had been a problem when
HEK293 cells were used (109). However, it did not produce enzyme of high
enough purity to enable in-depth kinetic investigation of the exopeptidase
activity, or any investigation at all into the endopeptidase activity.
One of the obstacles was that the heterologously expressed, histidinetagged enzyme did not appear to bind to the IMAC column (Paper II). This
resulted in low yield as well as low purification (Fig. 9). It was hypothesized
that the N-terminally positioned histidine tag was embedded in the complex,
thus unable to bind to the metal ions in the gel matrix. To improve binding, I
therefore attempted to dissociate the complex with guanidine, urea and increased pH, respectively. While the first two treatments increased binding
slightly, it was a very precarious balance not to denature the protein, since
renaturation through dialysis was impossible. In addition, while an increase
in pH to 8.5 did not destroy the enzyme, nor did it appear to increase binding
to the IMAC column or even dissociate the complex. Following these results, IMAC was abandoned as a purification strategy.
Since good results had been achieved using Escherichia coli as a host for
dTPP II (70), expression of mTPP II was commenced in this organism. An
27
initial expression system was set up in BL21(DE3) cells using a pET vector.
Subsequently, the first 200 base pairs were substituted for a version codon
optimised for E. coli, and the bacterial strain was exchanged for Rosetta
(DE3). However, the resulting expression was merely 5% of that achieved
for dTPP II, and neither optimization of codons nor expression conditions
increased expression yield more than marginally. It is possible that the
mRNA structure of mammalian TPP II hinders rapid translation. Nevertheless, the E. coli system provided higher yield and easier handling conditions
than the P. pastoris system previously used, and was thus utilized as the
starting point for the new purification system.
Unfortunately, the new expression system made binding to the anion exchange matrix difficult, since high nucleic acid content conferred high ionic
strength as well as high density. To rectify this, a protocol based on polyethylene imide and ammonium sulfate precipitations were developed that allowed subsequent binding and purification. Two additional chromatography
steps were added, hydrophobic interaction chromatography and gel filtration,
resulting in 80% or higher purity (Paper III). In all, this system conferred a
much higher yield and purity for wild-type mTPP II and mutants thereof
than any previously published procedure (Fig. 9).
Figure 9. Comparison between the two protocols used in this work, namely the
Pichia pastoris expression system combined with IMAC (Paper II) and the E. coli
expression system and three-step purification procedure described in Paper III.
28
How does the kinetic behaviour of TPP II vary with pH?
(Paper III)
To better understand the kinetic behaviour of TPP II, a pH dependence study
was undertaken, comparing mTPP II, hTPP II and dTPP II. The results revealed a difference in the pH-optimum profiles of two substrates differing
only in the P1 position, AAF-pNA or AAA-pNA. The suspotbstrate with
alanyl in the P1 position had a much flatter pH profile without any clear optima. This effect was surmised to stem from differences in the kcatdependence on pH (Paper III).
Throughout the investigation, it was obvious that the AAA-pNA substrate
yielded both lower KM and kcat than AAF-pNA. This could be caused by
non-productive binding, as the smaller substrate might have the possibility to
bind in more than one conformation to the active site, although only one
would be favourable for catalysis (Paper III).
At a pH above 7.6 there was an increase in KM with pH for all enzyme/substrate combinations (Fig. 10). The increase in KM at higher pH
could be due to deprotonation of the N-terminal amino group of the substrate. However, dTPP II had a minimum in KM for both substrates around
pH 7-7.5. It was hypothesized that the S1 pocket in dTPP II was compressed
by one or more protonated groups, as this would decrease the binding affinity at lower pH.
0.0
log KM
1.0
2.0
pH-dependence of KM
6.0
6.5
7.0 7.5
pH
8.0
8.5
Figure 10. The pH dependence of KM for mTPP II ( ) and dTPP II ( ) using AAFpNA as a substrate. Adapted from Paper III.
29
In a newly built homology model of mTPP II based on the crystal structure of dTPP II, it could be seen that the binding pocket for S1 was broad
enough to allow a small residue like alanine to bind in a less defined manner
than a bulkier one such as phenylalanine. It was hypothesised that His-267
(mTPP II numbering) in the vicinity of the catalytic triad could be responsible for the difference in pH-dependence between the two substrates. Furthermore, Asp-474 (dTPP II numbering), located behind the S1 pocket in
dTPP II but not mTPP II or hTPP II was identified as a potential candidate
responsible for the difference in pH-dependence in KM.
In conclusion, the studies of pH-dependent kinetic parameters have revealed differences in catalysis and substrate binding both between species and
between substrates. Finding the reasons behind these discrepancies would
provide valuable information on the substrate binding region of TPP II.
What is the substrate specificity of the endopeptidase
activity of TPP II? (Paper IV)
When the endopeptidase activity of TPP II was first discovered, it was proposed to favour basic residues in the P1 position of its substrates (69). That
would separate it from the exopeptidase activity, which preferentially
cleaves after aromatic or aliphatic amino acids (75). While it seemed plausible that the endopeptidase activity originated from the same active site as the
exopeptidase, this did not explain the apparent discrepancy in substrate
specificity.
Since only seven cleavage sites in two different peptides had been found
so far (69, 90), and little or no kinetic measurements had been carried out for
the endopeptidase activity, we decided to make a more thorough examination of this activity. As a starting point, we used the peptide Nef69-87, which
had been previously shown to be cleaved at two positions (90). Of these two
positions, only one could be verified, and several previously unreported
cleavage sites were found, both for the human and the murine enzymes (Paper IV). In addition to Nef69-87, the peptide LL37 was also used in the investigation, and was found to be cleaved at several positions, mostly after basic
residues. Taken in total, the results suggest that the endopeptidase activity is
less selective than previously reported (see Fig. 2 of Paper IV).
The rate of hydrolysis of the endopeptidase substrates were, as previously
reported, approximately four-five orders of magnitude lower than for the
exopeptidase activity. This increase could be due to a much lower binding
affinity, since the interaction with the N-terminal amino group reported in
Paper II is most likely lost. Cleavage experiments using the E331Q mutant
studied in Paper II could confirm this.
30
The specific TPP II inhibitor butabindide, which has a low-nanomolar KI
when measuring the exopeptidase activity, was not nearly as efficient at inhibiting the endopeptidase activity (Paper IV). Together with the discrepancies in substrate specificity, this suggests a different binding mode of the
substrate during endopeptidolytic cleavage. Butabindide was designed to
bind to the same amino acid residues that would interact with the N-terminal
amino group of the substrate during exopeptidolytic cleavage (101). This
interaction was later confirmed by our group (Paper II). If, during endopeptidolytic cleavage, the substrate is bound in such a way as to not interact with
these groups, it might evade this inhibition. In the homology model of
mTPP II based both on the crystal structure of dTPP II and subtilisin, the
substrate binding pocket is very broad, which explains the promiscuous substrate preferences, and possibly allows for an alternative substrate binding
mode during the endopeptidase activity (Fig. 8).
In conclusion, the endopeptidolytic substrate specificity does appear to be
different from the exopeptidase specificity, possibly due to a different mode
of binding. It also appears that the rate of endopeptidolytic hydrolysis is
four-five orders of magnitude lower than for the exopeptidase, which questions the significance of this activity under normal physiological conditions.
31
Conclusions
This work has provided several new insights into the structure and function
of TPP II. While it was previously known that TPP II existed in a broad
range of eukaryotic species, conserved sequence motifs have now been identified that can be used to find new homologues. The majority of these covered known regions of functional importance, and others have since been
noted to participate in interactions between monomers. We hope that these
signatures will facilitate more correct annotations of TPP II in the future.
The substrate binding mechanism of the exopeptidase activity has been
demonstrated to be dependent on Glu-331, a trait that is conserved between
TPP II homologues. Thus, a molecular ruler exists in TPP II, a phenomenon
that recurs in other peptidases cleaving peptides to a defined length.
Differences in the pH dependence of catalysis and substrate binding have
been revealed, both between species and between substrates. Further investigations into the reasons for these discrepancies will provide more detailed
information regarding the active site configuration in TPP II.
The substrate specificities seem to differ between the exo- and endopeptade activities, although the reason behind this could not be deduced in the
current investigation. No specific physiological substrate for the endopeptidase activity has been proposed, and the catalytic rate with the peptides used
in this investigation was very low. This argues against a role for the
endopeptidase activity under normal physiological conditions.
Variations in substrate binding dependent on pH, substrate, species or
exo- or endopeptidase activity have been investigated in this work. The image created is one of a flexible binding cleft, which allows substrates to bind
in modes incompatible with catalysis, or to ignore the molecular ruler
mechanism described above. This type of investigations should provide
valuable information for future drug development efforts, and facilitate the
determination of the role of TPP II in physiological functions.
32
Future perspectives
It is often the case that when a question is answered, several others appear as
were they curious onlookers of a spectacle. The present work makes no exception, as a multitude of future research topics arose from the observations
herein.
For example, some of the evolutionarily conserved sequence motifs may
provide excellent starting points for future investigations of the functions of
TPP II. Mutagenesis studies of single amino acid residues, e.g. one or both
of the conserved tryptophan residues would provide information regarding
the importance of these residues on substrate binding, catalysis or complex
formation. Since there have been reports of cellular functions being affected
by non-catalytic parts of the enzyme, it should prove worthwhile to test the
effects of these mutations in cell cultures a well.
The substrate binding mechanism of the exopeptidase activity, although
extensively studied, still holds some puzzles. Why is the binding of different
substrates affected dissimilarly by pH in dTPP II, compared to mammalian
TPP IIs? How can some substrates be bound in a non-productive manner?
The study of mutant variants of TPP II should provide some answers to these
questions, and thus provide further insights into the binding mechanism of
TPP II. This, in turn, would provide valuable information for future drug
discovery.
The endopeptidase activity poses an even greater enigma: why is the substrate specificity different between this and the exopeptidase activity? To
find an answer to this, the binding mode of endopeptidase substrates must be
elucidated, perhaps through a combination of docking studies and assays
with different peptide substrates. Furthermore, the kinetics of the endopeptidase activity is still virtually unexplored, and this would be a key element in
the clarification of the physiological role of this activity, perhaps through
endeavouring to create better FRET-substrates.
33
Sammanfattning på svenska
Syftet med det här avhandlingsarbetet är att utforska struktur och funktion
hos enzymet tripeptidylpeptidas II (TPP II), och hur samband mellan dessa
ger TPP II dess speciella egenskaper.
De flesta proteiner i cellen befinner sig i en jämvikt mellan nybildning
och nedbrytning. Cytosolisk nedbrytning av proteiner är en specifik process,
där proteiner märks med det lilla proteinet ubiquitin, vilket bildar långa kedjor som känns igen av 26S-proteasomen och bryts ned till peptider av olika
längd (se Fig. 5). Peptiderna måste sedan brytas ned helt till sina beståndsdelar, aminosyror, så att de kan återanvändas i nya proteiner. För att åstadkomma detta finns en hel uppsjö av peptidaser, som antingen kan klyva peptiderna från ändan (exopeptidaser) eller mitt i (endopeptidaser). Exopeptidaser kan antingen klyva från N-terminalen av peptider (aminopeptidaser) eller
från C-terminalen (karboxypeptidaser).
TPP II är ett av de peptidaser som hjälper till att finfördela peptider, vilket
den gör genom att klyva dem i tre aminosyror långa bitar. Dessa kan sedan
snabbt sönderdelas till fria aminosyror av andra aminopeptidaser. TPP II är
ett serinpeptidas, vilket betyder att den aktiva ytan har en katalytisk triad
bestående av aspartat, histidin och serin. Uppbyggnaden av TPP II är väldigt
speciell; peptidkedjan på 1249 aminosyror (hos däggdjur) veckas och bildar
dimerer, som sedan bildar långa strängar. Två strängar vrids sedan om varandra och bildar ett komplex, vars totala molekylvikt uppgår till 4-6 MDa, se
Fig. 6. Varför TPP II bildar ett så stort komplex är inte helt uppenbar, men
det står klart att den aktiva ytan ligger undangömd i ett grottsystem, dit inga
fullstora proteiner kan ta sig. Dimerer har en tiondel så hög aktivitet som det
fullstora komplexet. Det är möjligt att denna komplexbindning har selekterats fram som ett sätt att skydda cellen från oreglerad proteinnedbrytning.
TPP II förekommer hos alla djur och växter, och även i andra eukaryoter.
Den uttrycks i många olika vävnader, varför man har antagit att den har en
”hushållningsfunktion” i att snabba på peptidnedbrytningen. Utöver denna
funktion har TPP II även befunnits vara involverat i många andra av cellens
funktioner, såsom apoptos, mitos och antigenprocessering, och fysiologiska
processer som cancer, adipogenes och mättnadsrespons. Ännu vet man inte
vilken mekanism som ger dessa effekter, och mer information kan behöva
insamlas innan dess exakta funktion in vivo kan klarläggas. Eftersom TPP II
visats vara involverat i dessa processer, anses enzymet vara ett potentiellt
34
mål för läkemedelsutveckling, något som också kräver detaljerade kunskaper
om enzymets funktion och struktur.
Det första delarbetet (Paper I) beskriver konstruktionen av sekvenssignaturer baserade på väl konserverade regioner i peptidsekvensen hos TPP IIhomologer från många olika arter. Dessa signaturer är tänkta som redskap
för säkrare annotering av TPP II-homologer, men eftersom de är baserade på
delar av proteinet som är i hög grad konserverade, är de också förmodligen
regioner som har stor betydelse för struktur eller funktion. Av de elva signaturerna täckte sju redan kända funktionella eller strukturellt viktiga aminosyrarester, och av de övriga befinner sig två i gränsytan mellan aktiva domänen
och C-terminalen, något som kan vara strukturellt viktigt. Genom försök i
fissionsjäst, Schizosaccharomyces pombe, kunde vi visa att det enzym som
tidigare visats vara en funktionell homolog till TPP II också är en sekvenshomolog, vilket innebär att funktionella sekvenshomologer nu dokumenterats i både djur, växter och svamp. Under sökandet efter TPP II-homologer
hittade vi även ett bakteriellt enzym vars sekvens visade sig vara närbesläktat med TPP II från växter. Undersökningar visade att detta enzym troligen
hade tappat den TPP II-lika aktiviteten.
Exopeptidasaktiviteten hos TPP II klyver som sagt peptider i tre aminosyror långa bitar, från N-terminalen på substratpeptiden, som inte får vara
blockerad. En hypotes som undersöktes i det andra delarbetet (Paper II) var
att en eller flera negativt laddade grupper i TPP II interagerar med positivt
laddade N-terminalen på peptiden. På så sätt kan ett avstånd på tre aminosyror bildas till den katalytiskt aktiva gruppen, en ”molekylär linjal”. Från
sekvens- och strukturhomologi kunde två glutamatrester, Glu-305 och Glu331, utpekas som möjliga kandidater för den här interaktionen, och dessa
muterades båda två till glutamin och lysin. Från aktivitetsmätningar kunde
slutsatsen dras att bara en av de muterade varianterna, E331Q, hade tillräckligt hög aktivitet för att kinetiska parametrar skulle kunna bestämmas. Dessa
avslöjade att KM ökat åtminstone hundrafalt vid utbytandet av Glu-331 mot
en glutamatrest, vilket tyder på att denna är viktig för substratbindningen.
Ytterligare försök, gjorda med en peptidhämmare med och utan N-terminal
aminogrupp, visade på att E331Q band peptiden med och utan N-terminal
ungefär lika bra, medan det för vildtypsenzymet var en skillnad i IC50 på 2-3
tiopotenser.
Vidare undersökningar av exopeptidasaktiviteten har gjorts med avseende
på pH-beroende hos TPP II från tre olika arter och med två olika substrat,
AAF-pNA och AAA-pNA. Resultaten visade att ett av substraten, AAApNA, verkade kunna binda improduktivt till den aktiva ytan. Dessutom hade
detta substrat en plattare pH-profil, utan ett distinkt maximum, för alla enzymer. Detta berodde på en lägre ökning i kcat med pH p.g.a. deprotonering
av histidinet i den katalytiska triaden, vilket skulle kunna tyda på en negativ
effekt från en aminosyra med samma pKa. Varför denna effekt skulle påverka det ena substratet mer än det andra är i nuläget svårt att svara på. Den
35
största skillnaden mellan enzymen ligger i pH-beroendet av KM, där dTPP II
har ett tydligt minimum vid pH 7.6, medan de två mammala enzymen enbart
ökar i KM över pH 7.6, inte under. En hypotes som formulerades var att detta
berodde på en aspartatrest som bara återfinns i dTPP II.
Endopeptidasaktiviteten hos TPP II är mycket mindre välkaraktäriserad
än exopeptidasaktiviteten. I den sistnämnda finns en preferens för att klyva
efter aromatiska och alifatiska aminosyrarester, medan det har rapporterats
att endopeptidasaktiviteten klyver efter basiska aminosyrarester. Undersökningen som gjordes i delarbete fyra (Paper IV) kunde inte påvisa någon specifik preferens, och det verkar därför som om endopeptidasaktiviteten är mer
promiskuös än man tidigare trott. Hastigheten för hydrolys med endopeptidasaktiviteten är mycket lägre, omkring 4-5 magnituder, än den för exopeptidasaktiviteten. Detta gör att den fysiologiska relevansen för denna aktivitet
rimligen är låg, åtminstone i normalfallet. Det är dock möjligt att endopeptidasaktiviteten har betydligt högre affinitet för vissa naturliga substrat än för
dem vi undesökt.
Sammanfattningsvis har dessa undersökningar visat att det finns konserverade regioner i TPP II som är bevarade i alla eukaryoter, vilket förhoppningsvis ska göra TPP II-homologer lättare att hitta i framtiden. En av de
bäst bevarade regionerna innefattar Glu-331, som är viktig för bindning av
substrat, och utgör en ”molekylär linjal” i enzymet. För exopeptidasaktiviteten kunde även konstateras att vissa substrat kan binda på ett improduktivt
sätt, vilket tyder på att substratet kan binda på mer än ett sätt till den aktiva
ytan. Substratspecificiteten för endopeptidasaktiviteten kunde inte fastställas,
vilket till stor del berodde på den låga aktiviteten och en promiskuös tendens
hos enzymet.
36
Acknowledgements
Jag skulle vilja utsträcka ett stort tack till…/I would like to thank…
Birgitta, för att du alltid tagit dig tid, varit uppmuntrande och i största allmänhet varit en enastående handledare.
Helena, för att du är en outsinlig kraft- och inspirationskälla.
Mina medförfattare:
Ann-Christin, för tiden tillsammans på labb
Omar, keep on dancing like nobody’s watching.
Pernilla, för att du delade med dig av din jäst-expertis.
Hubert Kalbacher & crew, it was a pleasure working at your lab.
Emil, för den korta men otroligt intensiva tiden!
Micke, för att du delar med dig av din kunskap.
Exjobbarna genom åren: Linnea, Vanda, Mahmudur och Sabina, för tappra
insatser och goda skratt på labb.
Doktorander (och doktorer) på biokem:
Helena N (för sena nätter och tidiga morgnar i Grekland), Eric (för att du
stod ut med min värmefixering), Diana (för att du alltid är så positiv!), Natalia (för att du lärde mig hur man lär ut), Johan (det är alltid roligt att jobba
med dig), Angelica (för att du aldrig är rädd att fråga), Wei Zhang (I admire
your energy), Göran, Cissi, Sophia och alla nya och gamla biokemister!
Wei, for sharing office space and chitchat over the years!
Korridorskamraterna på IMBIM: Sophia, Pia, Åsa, Erik, Per, Chi, Raza,
Karin, Lisa, Andreas och alla andra som sett till att det varit liv och rörelse
på labb.
TA-personal på biokemi och IMBIM: Lillian, Inger, ”Nalle”, Barbro, Rehné,
Marianne, Erika och alla andra som alltid hjälper till!
Pappa, för att du alltid ställer upp och förstår allting…
Mamma, som alltid är entusiastisk och försöker sätta dig in i vad jag håller
på med.
37
Mina sedan-embryostadiet-vänner, Jossan och Ylva, för att ni finns där. Jossan speciellt för att du alltid är ärlig och rak och förväntar dig detsamma av
mig. Ylva, för att du alltid obevekligen är dig själv.
RotG-gruppen (Jonas, Magda, Fredrik och Toni, plus DD), för att ha gett
mina tankar annat att kretsa kring än labbet. Everyone speaks Giant!
Sist, men absolut inte minst, Lars, som försilvrar molnen på min himmel
varje dag.
Det här avhandlingsarbetet finansierades av Carl Tryggers Stiftelse, O.E. och
Edla Johanssons Vetenskapliga stiftelse, Kemistsamfundet och Liljewalchs
resestipendium
38
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