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The Effects of Cathepsin Inhibition on Trace
Amine-Associated Receptor 1 (TAAR1)
Localization
By: Zaina Batool
A thesis submitted in partial fulfillment of the requirements of a bachelor of science in
Biochemistry and Cell Biology
Jacobs University
Department of Life Sciences and Chemistry
15 May 2016
Matriculation Number: 20331001
520301 Guided Research Biochemistry and Cell Biology I
520302 Guided Research and BSc Thesis Biochemistry and Cell Biology II
1
TABLE OF CONTENTS
Abstract………………….……………………………………………………………………………...…..2
Introduction……….………....………………………………..……………………………………..……...2
Materials and Methods……....………………………………..…………………………………..………...4
Results……..……….………....………………………………..…………………………………………...7
Discussion………….………....…………………………………………………………………………...16
LAB ROTATIONS
520301 Guided Research Biochemistry and Cell Biology I
Performed by Zaina Batool in the laboratory of Prof. Dr. Klaudia Brix under supervision of Joanna
Szumska
Fall semester 2015
The Effects of Cathepsin Inhibition on Trace Amine-Associated Receptor 1 (TAAR1) Localization in
Nthy-Ori 3-1 Cells
520302 Guided Research Biochemistry and Cell Biology II
Performed by Zaina Batool in the laboratory of Prof. Dr. Klaudia Brix under supervision of Joanna
Szumska
Spring semester 2016
The Effects of Cathepsin Inhibition on Trace Amine-Associated Receptor 1 (TAAR1) Localization in
FRT Cells
2
Introduction
Thyroid function is dependent on the synthesis
and proteolytic degradation of thyroglobulin
(Tg). Nascent Tg is transported along the
secretory pathway to the apical plasma
membrane of thyroid follicular epithelial cells.
Following exocytosis, it undergoes storage in
covalently cross-linked globules within the
thyroid follicle lumen. Thyroid hormone (TH)
liberation occurs following the solubilization
and limited proteolysis of Tg in the thyroid
follicle lumen. This stage of Tg utilization is
followed by endocytosis and its lysosomal
degradation within thyroid epithelial cells [1, 2].
It has been shown that cathepsins B and L are
responsible for the solubilization of Tg, while
the utilization of luminal Tg for thyroxine (T4)
liberation is mediated by a combinatory action
of cathepsins K and L [2]. These cysteine
cathepsins belong to the papain-like family C1
of clan CA cysteine peptidases. Cathepsins B
and L are ubiquitously expressed in human
tissue while cathepsin K is highly expressed in
the ovary, osteoclasts and most epithelial cells
[19]. These peptidases exert their proteolytic
activity in the endocytic pathway, and following
secretion, in the pericellular space in
soluble or cell surface receptor-bound form [3,
18]. It has also been shown that stimulation of
thyroid stimulating hormone receptor (TSHR)-
bearing Fisher rat thyrocytes (FRTL-5)
correlates with a significant increase in
cathepsin B secretion, suggesting TSH
regulation of Tg processing in the extracellular
follicle lumen mediated by cathepsins [4].
Thyroid hormone regulation was previously
thought to occur following the passive diffusion
of thyroid hormones (TH) through the plasma
membrane (PM) of a target cell. This would
supposedly be followed by the intracellular
deiodination of thyroxine (T4) to triodothyronine
(T3), the nuclear entry of T3, and the binding of
The Effects of Cathepsin Inhibition on Trace Amine-Associated Receptor
(TAAR1) Localization
Performed by Zaina Batool in the laboratory of Prof. Dr. Klaudia Brix under the supervision of Joanna
Szumska
Abstract: The prohormone thyroglobulin (Tg) is subject to limited proteolysis by cysteine cathepsins to
yield thyroid hormones (THs). THs are then decarboxylated and deiodinated to generate thyronamines
(TAMs), which act as ligands to the trace amine-associated receptor (TAAR1). Recently, our group has
shown Taar1 to be localized in the primary cilium of thyroid epithelial cells in vitro and in situ. This
apical localization of Taar1 sheds light on the possibility that thyronamine biosynthesis could occur in the
thyroid follicle lumen. It is therefore hypothesized that the cilia of thyrocytes are involved in sensing the
amounts of extracellular formation of iodothyronines upon TH liberation through cathepsin-mediated Tg
degradation. We report reduced immunoreactivity of Taar1 at cilia of thyrocytes upon inhibition of
cathepsins B and L, and also observe an accumulation of monomeric Tg following inhibition of cathepsin
B. Moreover, there is increased Taar1 immunoreactivity at the endoplasmic reticulum of cultured
thyrocytes upon cathepsin L inhibition. Keeping in mind our group has shown colocalization of cathepsin
L and Taar1 at the cilia of FRT cells, we observed cilia formation in cathepsin-inhibitor treated cultures.
We report that inhibition of cathepsin B for 8 hours halts cilia formation in vitro. Our group has also
observed an increased incidence of dead cells in the follicular lumina of the thyroid of Taar1-deficient
mice. We sought to identify which form of programmed cell death (PCD) was responsible for this
phenotype and conclude apoptosis is less likely to be implicated than other forms of PCD, namely
necrosis and mitotic catastrophe.
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T3 to nuclear TH receptors, resulting in the
regulation of target genes. T3 exerts a genomic
effect by regulating the expression of target
genes and a non-genomic effect by activating the
PI-3 kinase signaling pathway [5, 6, 7].
However, this classical view of thyroid hormone
regulation has been revised with the discovery of
thyroid hormone transporters, such as the mono-
carboxylate transporters 8 and 10 (Mct8;
Mct10), the L-type amino acid transporter 2
(Lat2), and the organic anion-transporting
polypeptides (OATPs), which mediate the
cellular entry of THs [8, 9, 10, 11].
MCT8 belongs to the monocarboxylate
transporter family and mediates the transport of
aromatic amino acids, iodothyronines, and
amino acid derivatives. It is highly expressed in
the rat and human liver, kidney and brain [8].
Arguably, its most important role in the thyroid
is the cellular uptake and efflux of T3 and T4
[11]. L-type amino acid transporter 2 (Lat2) is a
sodium-dependent transporter of iodothyronines
and both small and large neutrally charged
amino acids ranging from alanine to tryptophan.
Lat2 also mediates the cellular entry of T3 [5,
10].
Thyronamines (TAMs) are endogenous amine
compounds generated by the decarboxylation
and deiodination of classical thyroid hormones
T3 and T4. Unlike their hormone precursors,
TAMs lack a carboxyl group on their β-alanine
side chain and differ in their states of iodination
[12]. The action and role of thyronamines is only
partially understood; however, they are believed
to be antagonists of classical THs. In one case,
the administration of synthetic 3-
iodothyronamine (3-T1AM) and thyronamine
(T0AM) resulted in dose-dependent bradycardia,
hypothermia, and hyperglycemia in mice; these
effects are opposites of the effects --
tachycardia, hyperthermia, and hypoglycemia --
of classical THs [12]. TAMs were consequently
termed as “cool” thyroid hormones [5].
3-idodothyronamine (3-T1AM) and thyronamine
(T0AM), a new class of ligands, have been
shown to bind to trace-associated amine receptor
1 (TAAR1) in rats (rTaar1), mice (mTaar1), and
humans (hTAAR1) [12, 13]. Trace-amine
associated receptors (TAARs) form a class of
rhodopsin-like G-protein coupled receptors
(GPCRs) which are activated by the binding of
trace amines such as p-tyramine, β-
phenylalanine, tryptamine and octopamine [14,
15]. They are highly conserved 7-
transmembrane domain receptors, with their N-
terminal domain positioned in the extracellular
space and the C-terminal domain located in the
cytosol. TAAR1 also contains a putative
disulfide bond connecting the second and third
extracellular loops of the receptor [16]. Human
Taar1 (hTAAR1) is comprised of 339 amino
acids, contains 2 potential N-glycosylation sites
at Asn residues, and has a molecular mass of
approximately 39.1 kDa. Mouse Taar 1
(mTaar1), meanwhile, has only one potential N-
glycosylation site and holds a molecular weight
of around 37.6 kDa [16]. TAARs have been
shown to be expressed in various human and
rodent tissues. In particular, TAAR1 is
expressed in various regions of the human,
rhesus monkey, and mouse central nervous
system (CNS) [14, 16].
Recently, the Brix group has shown Taar1 to be
localized in the primary cilium at the apical
plasma membrane domain of thyroid follicular
epithelial cells in mouse tissue and in cultured
Fisher rat thyroid cells [17]. This apical
localization of Taar1 sheds light on the
possibility that TAMs could be prevalent in the
thyroid follicle lumen. It was therefore
hypothesized that TAMs activate Taar1 in
thyroid follicular epithelia from within the
thyroid follicle lumen, their potential site of
synthesis, to serve as part of a non-classical
mechanism of thyroid auto-regulation [17]. The
postulated pathway for TAM derivation is by the
decarboxylation and deiodination of THs, which
are generated by the cathepsin-mediated
processing of the prohormone Tg. Interestingly,
another hypothesis postulates that 3-T1AM
undergoes intra-intestinal formation, a process
which is contingent upon the intestinal passage
of T4 [19].
Following this, we wanted to investigate the
effects of the inhibition of cathepsins B, K, and
L on TAAR1 localization bearing in mind that
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these proteases are important for TH liberation
by extra- and intracellular means of Tg
processing; THs act as TAM precursors. Our
hypothesis incorporated the proposal that a
change in the quantity of TAMs being generated
would affect TAAR1 expression and its
localization at the cell surface. To this end, we
performed the pharmacological knockdown of
the activity of cathepsins B and L in FRT
cultures and reported that this resulted in
reduced immunoreactivity of Taar1 on their
primary cilia. Moreover, there was increased
Taar1 immunoreactivity at the endoplasmic
reticulum (ER). To further explore this reduced
cilial localization of Taar1, we examined cilia
formation by using acetylated α-tubulin as a
marker of basal bodies, which are found at the
base of cilia. We questioned whether the
duration of incubation with cathepsin inhibitors
was a key factor in causing changes in Taar1
localization at cilia and thereby performed
cathepsin inhibition studies using short
incubation times, namely 4 hours and 8 hours.
Moreover, we took note that cathepsins and
Taar1 are transported along the secretory
pathway, diverging at the level of the trans golgi
network (TGN) where Taar1 continues to the
apical plasma membrane domain while
procathepsins are transported to late endosomes
for maturation and proteolytic activation [30].
Upon TSH stimulation, cathepsins are secreted
by thyrocytes and become re-associated with
plasma membrane and eventually re-internalized
at later time points. While FRT cells don’t bear
the TSH receptor, they are known to secrete
small amounts of cathepsins.
Interestingly, the Brix group has also found that
Taar1 was colocalized with cathepsin L at the
primary cilium in FRT cultures (unpublished
data). In order to unravel the relationship
between Taar1 trafficking and cathepsin L
expression, we studied the localization of Taar1
in cathepsin-deficient mice. It has been shown
that cathepsin L-deficient mice are characterized
by the incidence of dead cells in the follicular
lumina [2]. The Brix group, meanwhile, has also
demonstrated that Taar1-deficient mice exhibit
an increased incidence of dead cells in the
follicular lumina, particularly an incidence of
6% in Taar1-deficient mice compared to 3% in
wild type mice (unpublished data).
Programmed cell death (PCD) is the death of a
cell in any pathology when mediated by an
intracellular program, and refers to apoptosis,
autophagy, and programmed necrosis. These
three forms of PCD jointly decide the fate of
cells, while apoptosis and programmed necrosis
invariably affect cell turnover [29]. We therefore
performed cell death assays using caspase-3 as a
marker of apoptosis to identify which cell death
pathway led to the remnants of dead cells in the
lumina of the thyroid of Taar1-deficient mice.
Materials and Methods
Cell culture and cathepsin inhibition
The human thyroid follicular epithelial cell line
Nthy-Ori 3-1 and the rat thyrocytes FRT and
FRTL-5 were grown at 37 °C and 5% CO2 in a
moisturized atmosphere incubator (Heraeus
Instruments GmbH, Osterode, Germany). Nthy-
Ori 3-1 was grown in RPMI-1640 medium
including 10% Fetal Bovine Serum (FBS)
(Biowhittaker, Verviers, Belgium). FRT cells
were grown in Coons F-12 medium (Sigma-
Aldrich, Steinheim, Germany) containing 2.68
mg/mL sodium bicarbonate and 5% FBS
(F7524, Sigma-Aldrich Chemie GmbH,
Taufkirchen, Germany), supplemented with a
hormone mixture consisting of 2 μg/mL insulin
(I6634, Sigma-Aldrich), 20 ng/mL Gly-His-Lys
complex (G7387, Sigma-Aldrich), 5 μg/mL
transferrin (11107-018, Invitrogen, Darmstadt,
Germany) and 10 ng/mL somastostatin (S1763,
5
Sigma-Aldrich). Identical media were used for
FRTL-5 cells, with the addition of 1mU/mL of
TSH (T-8931, Sigma-Aldrich).
Cysteine cathepsins were inhibited by incubating
cultures with media containing the following
protease inhibitors for 16, 8, or 4 hours at 37°C,
5% CO2 in a moisturized atmosphere (Heraeus):
CA074 (205530, Calbiochem) targeting
cathepsin B, Cath L Inhibitor III (219427,
Calbiochem) of cathepsin L, Odanacatib
(MK0822, Selleckchem) specific for cathepsin
K, and E64 (BMLPI1070001, Enzo Life
Sciences) inhibiting all cysteine proteases. Their
concentrations can be found in the Appendix
(supplemental table 1).
RT-PCR
Total RNA was extracted using the RNeasy
Mini Extraction Kit (Qiagen, Hilden, Germany).
10 μg of RNA from each cell line was used as a
template for cDNA synthesis. Each reaction
contained 10 μg of RNA, 2 μL of reaction buffer
with MgCl2, 2 μL of dNTPs, 0.5 μL of random
oligonucleotide primers, and 3 μL of nuclease-
free water, and proceeded at 37°C for 60 min.
The cDNA for human TAAR1 was amplified
using the following primers: TAAR1 sense 5’-
ATGGTGAGATCTGCTGAGCA-3 and 5’-
TCCTCTGCAGTGAACATGTT-3’ antisense.
TAAR1 cDNA was amplified at an annealing
temperature of 60°C. Each PCR reaction
contained 0.5 μL of primers sense and
antisense, 1 μL of dNTPs, 5μL of Taq buffer +
KCl, 0.9 M MgCl2, 0.3 μL of Taq DNA
polymerase, and nuclease-free water in total
volume of 50 μL (Fermentas). The RT-PCR
products were separated through 1.5% agarose
gel electrophoresis and visualized by inclusion
of 0.3% ethidium bromide. FastRuler™ Low
Range DNA Ladder (SM1103, Thermo Fisher)
was used as a marker.
Indirect immunofluorescence and image
analysis
Cells used for indirect immunofluorescence
were cultured on cover slips in 6 well plates.
The cells were washed with CMF-PBS (calcium
and magnesium-free PBS, 0.15 M NaCl, 2.7
mM KCl, 1.5 mM NaH2PO4 x 2 H20, 8.1 mM
Na2HPO4, pH 7.4) 3 times for 5 min at 37 °C
and fixed with 4% paraformaldehyde (PFA) in
200 mM HEPES, pH 7.4 for 20 min at room
temperature. After washing cells thrice for 5 min
with both 200 mM HEPES and CMF-PBS at
room temperature, permeabilization with 0.2%
Triton X-100 or 0.2% saponin in CMF-PBS was
performed for 5 min at room temperature,
followed by 4 washes in CMF-PBS lasting 5
min each. The blocking was done with 3%
Bovine Serum Albumin (BSA; Carl Roth
GmbH, Karlsruhe, Germany) in CMF-PBS for 1
hour at 37 °C, after which the cells were washed
twice for 5 min in 0.1% BSA in CMF-PBS.
Incubation with primary antibody overnight at 4
°C was done with polyclonal goat anti-mouse
DPP4 (1: 200; R&D Systems), polyclonal rabbit
anti-human APN (1:200; SC15360, Santa Cruz
Biotechnologies), polyclonal mouse anti-human
Lamp2 (1:50; H4B4, DSHB), and polyclonal
mouse anti-human Golgin97 (1:50; A-21270,
Molecular Probes), polyclonal rabbit anti-mouse
Taar1 (1:30; ABIN352010, Antibodies Online),
rabbit anti-active caspase-3 (1: 30, 559565, BD
Pharmingen), and mouse anti-rat acetylated α-
tubulin (1:30, T7451, Sigma Aldrich).
Afterwards, the cells were washed 6 times 5 min
with 0.1% BSA in CMF-PBS at room
temperature and incubated with Alexa 488-
conjugated or Alexa 546-conjugated secondary
antibodies with the addition of 5 mM DRAQ5™
(1:5000; Biostatus Ltd., Shepshed
Leicestershire, UK) for 60 min at 37 °C. After
washing the cells thrice for 5 min with CMF-
PBS and once for 1 min with ddH2O the cover
slips were mounted onto glass microscopy slides
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using embedding medium (33% glycerol, 14%
Mowiol in 200 mM Tris-HCl, pH 8.5, Hoechst,
Frankfurt, Germany). The same procedure was
used for immunostaining with biotin-conjugated
Concanavalin A (1:200; SLBC2190V, Sigma-
Aldrich, Munich, Germany) followed by Alexa
546-conjugated Streptavidin (1:5000; S11225,
Molecular Probes). Microscopy was done with a
confocal laser scanning microscope (LSM 510
Meta; Carl Zeiss Jena GmbH, Jena, Germany)
and the analysis was done with the LSM 510
software, release 3.2 (Carl Zeiss Jena GmbH).
Indirect immunofluorescence was also
performed on thyroid tissue. Cryo-sectioning of
the thyroid was performed at 5 µm after
embedding and freezing of the thyroid in tissue
freezing medium (-20ºC, 14020108926, Leica
Biosystems, Nussloch, Gerrmany). The sections
were then transferred to positively charged
slides and kept at -20º C until used. Tissue was
then blocked in 3% Bovine Serum Albumin
(BSA) in CMF-PBS for 1 hour at 37º C. The
tissue was then washed 3 times for 5 minutes in
0.1% BSA in CMF-PBS after which the primary
antibody was incubated overnight at 4º C. The
primary antibody used was rabbit anti-mouse
Taar1 (1:60, HPA055614, Atlas Antibodies,
Sigma-Aldrich). Following this, 6 washes were
performed for 5 minutes with 0.1% BSA CMF-
PBS at room temperature and then incubated
with secondary antibodies for 1 hour at 37º C.
The secondary antibody used was goat anti-
rabbit Alexa Fluor 488 (1:200) with the addition
of 5 mM Draq 5 (1:500, Biostatus). The sections
were then washed 3 times for 5 minutes in CMF-
PBS and mounted with the use of mowiol,
covered with cover slips.
Immunofluorescence was observed with a
confocal laser scanning microscope (LSM 510
Meta; Carl Zeiss Jena GmbH, Jena, Germany)
and the analysis was done with LSM 510
software release 3.2 (Carl Zeiss Jena GmbH)
and the open source image analysis software
CellProfiler v2.1.1.
Protein isolation, SDS-PAGE and
immunoblotting
Cells were cultured in Petri dishes (100 mm
diameter, Sigma-Aldrich) in 7 mL of growth
media including protease inhibitors for 16h or
8h. They were then washed three times with
CMF-PBS (5 mL/dish) and scraped off with a
rubber policeman (Sarstedt) in 3 mL CMF-PBS.
The cell suspensions were collected in
microtubes and pelleted by centrifugation at 110
g at 4°C for 5 min. Pellets were resuspended in
200 µL lysis buffer at pH 7.4 containing 50 mM
Tris, 5 mM MgCl2, 2 mM DTT, and 0.5% Triton
X-100, supplemented with 5µM biotinylated
DCG-04 (from Matt Bogyo), and incubated for
30 min at 4 °C on a vertical rotor [26].
Following centrifugation at 16000 g for 10 min
at 4 °C, the supernatants containing activity-
based probe (ABP)-bound proteins were stored
at -20°C.
The Neuhoff assay was used to determine
protein concentrations [21]. The proteins (15
μg/lane) were separated on 12.5%
polyacrylamide gels (or 7% polyacrylamide gels
when Tg degradation fragments were analyzed).
Their sizes were determined using the PageRuler
Prestained Protein ladder (MBI Fermentas, St.
Leon-Rot, Germany). Proteins were then
transferred to Whatman®
nitrocellulose
membrane (Whatman, Dassel, Germany) using
the semi-dry transfer approach (transfer buffer:
(48 mM Tris, 39 mM Glycin, 1.3 mM SDS, 20%
methanol).). Unspecific binding of antibodies
was blocked by incubating the membrane with
5% milk powder in PBS-T (68 mM NaCl, 63.2
mM Na2HPO4, 11.7 mM NaH2PO4, pH 7.2,
0.3% Tween) overnight at 4°C. Incubation with
primary antibodies (1:500 in PBS-T) was
performed at room temperature for 2h with
7
monoclonal rabbit anti-bovine thyroglobulin
(1:500; Wolfgang Summa), goat anti-mouse
cathepsin B (1:500, GT15047, Neuromics), goat
anti-mouse cathepsin L (1:500, GT15049,
Neuromics), and polyclonal rabbit anti-human ß-
tubulin (1:500; ab-6046-100, Abcam,
Cambridge, UK). Incubation with horseradish
peroxidase-conjugated secondary antibodies
(1:200) was done at room temperature for 1h,
followed by incubation with horseradish
peroxidase substrate (ThermoScientific,
Germany) and visualization through enhanced
chemiluminscence on XPosure films
(ThermoScientific). Densitometry analysis of
immunoblots was performed using the open
source program ImageJ v1.49. DCG04 labeled
proteins were visualized by incubation of the
blots for 1 hour at room temperature with
horseradish peroxidase-conjucated streptavidin
(1:500) (DIANOVA, Hamburg, Germany).
Activity-based probes
Active cathepsins were visualized by incubating
cultivated cells in serum-free media containing
DCG-04 Red (1:1000; Matt Bogyo) or NS-173
Rhodamine (1:10000; Norbert Schaschke) for 30
minutes under the given culture conditions in 6
well plates containing coverslips [26, 27]. The
coverslips were washed three times for 1 min
with PBS and incubated with fresh media for 30
min. DRAQ5 (1:5000; Biostatus) was applied
for 20 min at room temperature after fixation
and the coverslips were washed 6 times for 1
min prior to mounting.
Results
Determining polarity of Nthy-Ori 3-1 cells
Dipeptidylpeptidase 4 (DPP4) and
(Aminopeptidase N) APN are both apical
plasma membrane domain markers [18].
Investigating whether Nthy-Ori 3-1 cells exhibit
epithelial polarity, we used immunofluorescence
to determine the localization of these markers
and found that they were dispersed on structures
throughout the cytoplasm instead of being
localized at the plasma membrane, which would
have indicated structural differentiation (Figure
1A-C). The Nthy-Ori 3-1 cells grew on top of
each other when cultured for a long time (>4
days) and exhibited a flat appearance.
TAAR1 expression and localization in Nthy-Ori
3-1 Cells
The expression of TAAR1 in Nthy-Ori 3-1 was
proven by using RT-PCR. The expected product
size of 300 bps was amplified from cDNA
template. No bands were present in the controls:
template only, reverse transcriptase only, and
water only (Figure 1J).
After confirming the expression of TAAR1 in
this cell line, we performed indirect
immunofluorescence to determine the
subcellular localization of the protein. A rabbit
anti-mouse Taar1 antibody was used to visualize
TAAR1. We used the following stainings as
compartment markers: a mouse anti-human
Golgin97 antibody specific against the golgi
apparatus (Golgi), and a mouse anti-human
LAMP2 antibody specific against lysosomes.
Additonally we used a lectin that binds α-D-
mannosyl and α-D-glucosyl groups of
glycoproteins and glycolipids residing at the
plasma membrane (PM), namely, biotinylated
concanavalin A, visualized by streptavidin
conjugated with a fluorophore. No emdoplasmic
reticulum (ER) marker could be used as of yet
due to limitations imposed by species-specificity
in the available collection of antibodies.
TAAR1 was seen to have reticular localization
throughout the cells, indicating its possible
localization within the ER. It was also seen to be
localized at the PM in every instance of reticular
localization; the ER and PM will be considered
as the same category in this study. In some rare
8
Figure 2: The effects of cathepsin inhibition on TAAR1 localization and Tg processing in human thyrocytes. A: Labelling of active
cysteine cathepsins with activity based probes in Nthy-Ori 3-1 cells treated with cathepsin inhibitors. B. Degradation fragments of Tg
in Nthy-Ori 3-1 cells treated with cathepsin inhibitors. Molecular mass markers are indicated in the left margins. Proteins were
separated by SDS-PAGE and immunoblotted with specific antibodies and secondary, peroxidase-coupled antibodies before
visualization through enhanced chemiluminescence. C: The subcellular localization of TAAR1 in Nthy-Ori 3-1 cells treated with
cathepsin inhibitors. *N indicates number of cells analyzed.
Figure 1: Epithelial polarity and TAAR1 expression in Nthy-Ori 3-1 cells. A: APN in Nthy-Ori 3-1 cells. Fluorescence channels merged
in left panel (APN in green, DRAQ5 in blue) and in right panel: top APN, middle DRAQ5, bottom phase. B: DPP4 in Nthy-Ori 3-1 cells.
Fluorescence channels merged in left panel (DPP4 in green, DRAQ5 in blue) and in right panel: top DPP4, middle DRAQ5, bottom
phase. C: DPP4 in Nthy-Ori 3-1 cells. Xz stack. Fluorescence channels merged in left panel (DPP4 in green, DRAQ5 in blue). D: TAAR1
in Nthy-Ori 3-1 cells. Single channel fluorescence micrographs in right panel: top TAAR1, middle DRAQ5, bottom phase contrast.
Fluorescence channels merged in left panel: TAAR1 in green and DRAQ5 in blue. E-H: Fluorescence channels merged: TAAR1 in
green, DRAQ5 in blue, and in red, Golgi Apparatus (E), PM (G) and Lysosomes (H). I: TAAR1 in Nthy-Ori 3-1 cells. Xz stack.
Fluorescence channels merged in left panel (TAAR1 in green, DRAQ5 in blue, PM in red). Scale bars 20 µm.J: Expression of TAAR1
mRNA in Nthy-Ori 3-1 cell; RT-PCR amplification of TAAR1 mRNA. Amplicons were analyzed by agarose gel electrophoresis.
Molecular size markers shown in the left margin.
9
cases, TAAR1 was localized in the Golgi but
was never observed in endolysosome-
resembling vesicles (Figure 1D-I).
Effects of cathepsin inhibition on Tg processing
and TAAR1 localization in Nthy-Ori 3-1 cells
Various inhibitors were used to suppress the
activity of cysteine proteases in Nthy-Ori 3-1
cells. Active cysteine cathepsins were labeled
using activity-based probes and the signal for
active cysteine cathepsins was seen to diminish
in the lysates of inhibitor-treated cells (Figure
2A). A Tg blot was performed and the signal for
monomeric Tg seemed to be stronger in the
lysates of cathepsins B, K, and L inhibitor-
treated cells than the lysates of E64-treated cells
(Figure 2B). However, the normalization of the
blot to β tubulin indicated unequal loading of
samples, so the change in the Tg signal was
discredited.
Subsequently, TAAR1 was visualized and its
subcellular localization was determined through
analyzing immunofluorescence micrographs.
The compartments taken into consideration were
the golgi apparatus and plasma membrane. The
subcellular localization of TAAR1 was
calculated as a percentage of total prevalence.
This quantitative analysis of
immunofluorescence micrographs was done
using Cell Profiler v2.1.1. The calculations
involved in this analysis are given in the
Appendix.
TAAR1 was always localized at the ER and PM
in untreated cultures; Nthy-Ori 3-1 cells do not
display heterogeneity of expression of TAAR1.
Upon inhibitor-treatment, TAAR1 was found in
the Golgi Apparatus in several cells per
treatment; however this change was not
significant (Figure 2C).
Labelling of active cysteine cathepsins with
biotinylated or fluorescently labeled activity
based probes in the FRTL-5 cells
After studying the subcellular localization of
TAAR1 in human thyrocytes, we wanted to
investigate the same phenomenon in Fischer rat
thyrocytes, namely, FRTL-5 cells. A blot was
obtained to visualize the cathepsins which had
reacted with DCG-04 biotin during lysate
preparation.
Once again, the most prominent band was
around the size of 27 kDa, indicating the
presence of active cathepsin B single chain
(Figure 1M). Upon cathepsin B-inhibitor
treatment, this band disappeared, suggesting
successful inhibition of this protease. No change
was observed following the cathepsin k-inhibitor
treatment, while the signal for active cathepsins
diminished in the lysates of cathepsin L-
inhibitor and E64 treated cells; the effect was
more prominent in the latter.
As additional controls of cathepsin activity
inhibition, we performed activity-based protein
profiling using the fluorescent probes DCG-04
red and NS-173 Rhodamine.
Using DCG-04 red, which is a fluorophore-
conjugated activity based probe for cysteine
cathepsins, signals for active cysteine cathepsins
were detected in the untreated cultures. Upon
inhibitor treatments targeting cathepsins B, K,
and L, these signals diminished. (Figure 3A-D)
The effect was most prominent upon the
inhibition of cathepsin B. Using NS-173
Rhodamine, which is a cathepsin B-specific
ABP, signals for active cathepsin B were
detected in untreated cultures and cathepsin K or
-L-inhibtor treated cultures. These signals
disappeared upon the cathepsin B-inhibitor
treatment of cultures (Figure 3E-H).
Next, we performed a Tg blot to report the
effects of cathepsin inhibition on Tg processing.
As accumulation of monomeric Tg (and some
degradation fragments) was observed in the
inhibitor-treated cell lysates; this effect was
10
Figure 3: Cathepsin Activity in FRTL-5 cells treated with cathepsin inhibitors. A-D: DCG-04 Red. E-H: NS-173 Rhodamine. Scale
bar: 20 μm.
Figure 4: Inhibition of cysteine cathepsins in FRTL-5 cells. A: Labelling of active cysteine cathepsins with activity based probes
in FRTL-5 cells treated with cathepsin inhibitors. Molecular mass markers indicated in the left margins. B: Degradation
fragments of Tg in FRTL-5 cells treated with cathepsin inhibitors. Molecular mass markers are indicated in the left margins.
Proteins were separated by SDS-PAGE and immunoblotted with specific antibodies and secondary, peroxidase-coupled
antibodies before visualization through enhanced chemiluminescence. C: Monomeric Tg in Cathepsin Inhibitor-treated FRTL-5
11
most prominent in the cathepsin B-inhibitor
treated cells, which was confirmed by optical
densitometry analysis (Figure 4C).
Effects of cathepsin inhibition on Taar1
localization in Fischer Rat thyrocytes
To this end, we first performed RT-PCR to
confirm the expression of Taar1, the results of
which are given in the appendix (Supplemental
Figure 1). Quantitative analysis of
immunofluorescence micrographs using
CellProfiler then yielded results for the
subcellular localization of Taar1 in FRT and
FRTL-5 cells. FRT cells displayed the greatest
heterogeneity of expression of Taar1 amongst
the cell lines used in this study; Taar1 was
localized in the Golgi, ER, or primary cilium of
these cells. Slight but significant reduction of
Taar1 immunoreactivity in primary cilia can be
observed in cathepsin B and L inhibitor treated
FRT cells.
The reductionof Taar1 immunoreactivity in
primary cilia of cathepsin L inhibitor-treated
FRT cells corresponded with a significant
increase in localization within the ER. (Figure
5A).
Analysis of immunofluorescence micrographs of
FRTL-5 cells revealed that Taar1 was abundant
at the PM while there was some localization
within the Golgi. As in human thyrocytes, in
instances where Taar1 was observed at the PM,
it was also found to be localized within the ER,
so these were considered as one category in
these localization studies.
There was no significant effect on Taar1
localization upon the inhibition of cathepsins in
FRTL-5 cells (Figure 5B).
Cathepsin expression in cathepsin inhibitor-
treated FRTL-5 cultures
Figure 5: Effects of cathepsin inhibition on Taar1 localization in FRT cells. A) The subcellular localization of Taar1 in FRT cells treated
with cathepsin inhibitors in ER (arrows), Golgi (asterisks), and cilia (arrowheads). B) The subcellular localization of Taar1 in FRTL-5
cells treated with cathepsin inhibitors in ER and PM (arrows) and Golgi (asterisks). Scale bar 20 μm. *N indicates number of cells
analyzed.
12
Our studies thus far have shown slightly reduced
Taar1 localization at the plasma membrane of
FRTL-5 cells upon the inhibition of cathepsin L;
however, through the use of activity-based
probes, we observed that cathepsin L was not
fully inhibited. Therefore, we performed a
cathepsin L immunoblot to determine whether
there was an upregulation of cathepsin L
expression subsequent to the inhibitor treatment.
When incubated with cathepsin inhibitors for 16
hours, FRTL-5 cultures showed increased
expression of the mature form of cathepsin L in
all treatments compared to the untreated control.
This change was most noticeable in the
cathepsin L inhibitor-treated culture. (Figure 6).
We then performed immunoblotting to
determine the expression levels of cathepsin L
after 8 hours of incubation with protease
Figure 6: A) Cathepsin L expression in FRTL-5 cultures
incubated with cathepsin inhibitors for 16 hours. Molecular
mass markers are indicated in the left margins. B)
Densitometry analysis of expression of cathepsin L mature
form in FRTL-5 cultures treated with cathepsin inhibitors.
Figure 7: A) Cathepsin L expression in FRT cultures incubated with cathepsin inhibitors for 8 hours. Molecular mass markers are
indicated in the left margins. B) Densitometry analysis of mature cathepsin L expression in FRT cultures incubated with cathepsin
inhibitors for 8 hours. C) Labelling of active cathepsins in cathepsin inhibitor-treated FRT cultures. D) Cathepsin B expression in
FRT cell cultures incubated with cathepsin inhibitors for 8 hours.
13
Figure 8: Basal bodies in FRT cultures incubated with cathepsin inhibitors for 4 hours. All treatments were performed in duplicates.
Fluorescence channels merged in left panel (Acetylated α-tubulin in green, DRAQ5 in blue) and in right panel: top acetylated α-tubulin, middle
DRAQ5, bottom phase. Scale bars: 10 μm.
Figure 9: Basal bodies in FRT cultures incubated with cathepsin inhibitors for 8 hours. All treatments were performed in duplicates.
Fluorescence channels merged in left panel (Acetylated α-tubulin in green, DRAQ5 in blue) and in right panel: top acetylated α-tubulin,
middle DRAQ5, bottom phase contrast. Scale bars: 10 μm..
14
inhibitors, now using the FRT cell line (Figure
7A). The immunoblot revealed the expression of
procathepsin L following all treatments and in
the untreated control, and the absence of
expression of mature cathepsin L in all cultures
except the cathepsin B- and K-inhibitor treated
ones. Furthermore, the intensity of the mature
cathepsin L-positive signal decreased in the
cathepsin L-inhibitor treated cultures
We analyzed the intensity of the mature
cathepsin L-positive signal through optical
densitometry and confirmed its absence in
cathepsin B- and K-inhibitor treated cultures and
its reduced expression in cathepsin L-inhibitor
and E64 treated cultures compared to untreated
cultures (Figure 7B). A blot was also obtained as
a control to visualize active cathepsins which
had reacted with DCG-04 biotin during lysate
preparation. A band corresponding to around 40
kDa was observed.
We also conducted immunoblotting to determine
the expression levels of cathepsin B after 8
hours of incubation with protease inhibitors
(Figure 7D). Unfortunately, the results from this
immunoblot are not conclusive – perhaps due to
the unspecific action of the cathepsin B
antibody.
Cilia formation in cathepsin inhibitor-treated
FRT cultures
We observed cilia formation by using the basal
body marker acetylated α-tubulin, in FRT
cultures in duplicates upon incubation with
cathepsin inhibitors for 4 and 8 hours. Ring-like
acetylated α-tubulin positive structures were
observed through immunofluorescence and were
identified as the basal bodies which are
characteristic of cilia-bearing cells.
The acetylated α-tubulin positive structures were
found in both duplicates across untreated
cultures and inhibitor-treated cultures when
incubated for 4 hours; no change was observed
in the formation of cilia upon 4-hour long
inhibition of cathepsins (Figure 8).
Consequent to the incubation of FRT cultures
with cathepsin inhibitors for 8 hours, we
reported an absence of acetylated α-tubulin
positive ring structures in both duplicates of
cathepsin B-inhibitor treated cultures (Figure 9).
Furthermore, the acetylated α-tubulin positive
ring structures were only found in one of the two
duplicates in cathepsin K- and L-inhibitor
treated cultures, while the other sample yielded
no change compared to the untreated cultures.
As we were expecting the same results to be
reflected in both duplicates, these results must
be declared inconclusive at present.
Surprisingly, no change was subsequent to the
E64 treatment, which inhibits all cysteine
proteases.
Figure 10: Taar1 in thyroid tissue of wild type and cathepsin-
deficient mice as indicated. Fluorescence channels merged from
Taar1 in green and DRAQ5 in blue. Stars indicate lumina, and
arrows point to Taar1-positive structures. Scale bars: 10 μm.
15
Taar1 localization in cathepsin deficient mice
Taar1 immunoreactivity was previously
demonstrated by our group at the apical plasma
membrane of thyroid follicles in wild type mice
[17].
We checked for immunoreactivity of Taar1 in
vivo in several cathepsin-deficient mice and
reported the following: there was no change in
Taar1 immunoreactivity in cathepsin B-deficient
mice compared to the wild type; there was no
Taar1 positive signal at the apical plasma
membrane in cathepsin K-deficient mice; Taar1
immunoreactivity was observed in reticular
structures intracellularly in cathepsin L-deficient
mice; and cathepsin B- and K-double deficient
mice displayed no Taar1 immunoreactivity
(Figure 10).
Cell death in Taar1 deficient mice
We used activated procaspase 3 as a cell death
marker in Taar1-deficient mice to compare the
incidence of apoptotic cell death with wild type
mice (Figure 11). Qualitative analysis of the
overview of thyroid lobes showed that there was
a slightly higher incidence of the caspase 3-
positive signal in the taar1-deficient mice. This
must be further confirmed with analysis of
several thyroid lobes from each genotype. At
present, we conclude that the faintness and
scarcity of the caspase 3-positive signal in the
Taar1-deficient mice does not indicate a higher
incidence of apoptotic cell death in Taar1-
deficient mice. Nonetheless, remnants of dead
cells were observed within the thyroid lumina of
the Taar1-deficient mice and could be indicative
of other cell death pathways.
Figure 11: Overview of thyroid gland in wild type (left) and Taar1-deficient (right) mice. Fluorescence channels merged (Cas3, DRAQ5)
16
Discussion
Nthy-Ori 3-1 cells do not exhibit epithelial
polarity
In the course of this study, we determined that
Nthy-Ori 3-1 cells do not exhibit signs of
epithelial polarity. This raises the question
whether this cell line is differentiated, which can
be determined through further studies
investigating the prevalence of transcription
factors such as TTF1 and Pax 8, which are
essential for the expression of proteins such as
sodium iodide symporter (NIS), TSH receptor,
Tg, and thyroperoxidase (TPO), which are
expressed in fully differentiated thyrocytes [20].
Resultantly, we discontinued our studies with
this cell line and instead focused on the effects
of cathepsin inhibition on Taar1 localization in
rat thyrocytes (FRT and FRTL-5).
TAAR1 localization in human and rat thyrocytes
TAAR1 is most abundantly located at the
plasma membrane in Nthy-Ori 3-1 and FRTL-5
cells (Figures 1L, 3D). These results are in
keeping with the findings of our group, where it
was concluded that Taar1 is transported along
the secretory pathway and accumulates in the
primary cilium of thyrocytes in rodents [17].
However, this is in contrast to previous studies
conducted with human embryonic kidney (HEK
293) cells in which heterologously expressed
Taar1 was found to be localized mostly
intracellular [15]. This could be due to
differences in Taar1 trafficking between
thyrocytes and HEK239 cells, and due to
differences in trafficking of heterologously
expressed TAAR1 compared to endogenous
TAAR1; the latter was the focus of our studies.
Meanwhile, Taar1’s localization at the primary
cilium of FRT cells (Figure 3C) is intuitive since
this was the only structurally differentiated cell
line displaying cilia used in this study.
The effects of cathepsin inhibition on Tg
processing and TAAR1 localization in human
and rat thyrocytes
The inhibition of cysteine cathepsins was
successful in Nthy-Ori 3-1 cells as the signals
for these proteases diminished compared to the
untreated lysates. However, the inhibitors used
might not be specific for cathepsins B, K, and L,
as these inhibitors lead to the disappearance of
the same bands as E64, which is an inhibitor for
all cysteine proteases (Figure 1K). Furthermore,
this did not have an effect on Tg processing
(Figure 2A). This could be due to the
compensatory action of other proteases with Tg
processing ability. Indeed, previous studies have
shown aspartic cathepsin D to compensate for a
lack of cathepsin L and cathepsin L to
compensate for a lack of the proteolytic activity
of cathepsin K [2].
Upon the inhibition of cysteine cathepsins in
Nthy-Ori 3-1 cells, some TAAR1 localization
was observed within the golgi apparatus (Figure
Figure 12: Schematic representation of the putative location of
TAM generation and contribution to autoregulation of the
thyroid gland. Tg degredation is mediated by cysteine
cathepsins B, K, and L, starting in the extraceullular follicle
lumen and proceeding in the compartments of the endocytic
pathway [2].
17
2C). This change was not significant and further
studies had to be conducted to establish the
prevalence and cause of this phenomenon. The
inhibition of cathepsin B was successful in
FRTL-5 cells, as the signal for a band of 27 kDa
which corresponds to the size of the mature form
of this protease disappeared upon inhibitor
treatment (Figure 4B). This finding is supported
by our controls; the fluorescently labeled ABPs
DCG-04 red and NS-173 Rhodamine also
indicated the success of the inhibition of
cathepsin B (Figure 3). This was also reflected
in the accumulation of monomeric Tg (330 kDa)
in cathepsin B inhibitor-treated cultures,
indicating that the proteolytic function of
cathepsin B was suppressed. Despite this, there
were no significant changes in Taar1
localization in FRTL-5 cells (Figure 5B).
The inhibition of cathepsins B and L led to
reduced Taar1 localization at the primary cilia of
FRT cells (Figure 5A). The cause behind
reduced localization at the cilia of cathepsin B
inhibitor-treated cultures remains to be
investigated. Cathepsin L was also shown to be
localized at the primary cilium in previous
studies [18]. Additionally, it was suggested that
cathepsin L plays a crucial role in the survival of
thyrocytes [2]. Our study was then repeated with
short incubation times in efforts to unravel the
relationship between Taar1 and the suggested
role of cathepsin L in thyrocyte survival, Tg
processing, and follicle maturation, and to gain
better insight into thyroid metabolism and
function.
The effects of cathepsin inhibition on cilia
formation in Fischer rat thyrocytes
As our previous studies had shown a change in
Taar1 trafficking upon the pharmacological
knockdown of cysteine cathepsin activities, we
wanted to explore the possible mechanisms
behind Taar1’s reduced cilial localization.
Keeping in mind that Taar1 and cathepsin L had
been colocalized at the primary cilia of FRT
cells (unpublished data), we conducted studies to
determine whether cilia formation was affected
by inhibition of the activity of cysteine
cathepsins using specific and broad-spectrum
inhibitors. We found that cilia formation was
unaffected by 4 hour-long incubations, and that
an 8 hour-long incubation with a cathepsin B
inhibitor halts the formation of cilia. The
mechanism behind cathepsin B’s interference
with cilia formation remains uncertain.
Moreover, the effects of 8 hour-long incubations
with inhibitors of cathepsins K and L are
inconclusive at present. The studies must be
repeated with a larger number of replicates and
quantitative analysis must be performed to
determine whether there is a significant change
in the cilia-bearing nature of cathepsin inhibitor-
treated FRT cultures.
It is of note that one possible mechanism
contributing to increased Taar1 localization to
ER could be attributed to an upregulation of the
de novo biosynthesis of Taar1. This could be
verified by blocking the de novo synthesis of all
proteins prior to the inhibition treatment using
cycloheximide, a known inhibitor of ribosomal
function [28], and then determining Taar1
localization using immunofluorescence.
Another route of suggested future investigations
involve checking the Taar1 mRNA and of
protein levels. Furthermore, activity assays must
be conducted to control for the inhibition of the
activity of cathepsins. Our cathepsin B
immunoblot was unsuccessful, but our cathepsin
L immunoblot showed increased expression of
the cathepsin L heavy chain in FRTL-5 cultures
after 16 hours of incubation with all cathepsin
inhibitor-treatments (Figure 6).
18
Cell death in Taar1 deficient mice
Knocking out Taar1 and cathepsin L resulted in
the increased incidence of remnants of dead cells
in thyroid lumina.
Cathepsin B has previously been implicated in a
lysosomal pathway to apoptosis [31]. Inducing
lysosomal permeabilization causes efflux of
cathepsin B into the cytosol, where cathepsin B
cleaves the proapototic member of the Bcl-2
family, Bid, thereby inducing the intrinsic
pathway of apoptosis. This is followed by the
release of cytochrome c (Cyt c) from
mitochondria, apoptosome formation, and
activation of caspase-9 and -3 [31].
We conducted an investigation into whether cell
death occurs through apoptosis in Taar1-
deficient mice, but qualitative analysis led to the
conclusion that this is not the case (Figure 10)
because procaspase-3 activation occurred to
comparable extents in both wild type and Taar1-
deficient thyroid tissue.
Further studies must be conducted to determine
the mechanisms leading to cell death, which can,
in principle, be apoptotic, necrotic, autophagic,
or associated with mitotic catastrophe [29].
Given the interdependence of Taar1 and
cathepsins at cilia, the latter option in particular
is suggested for future experimentation;
observing microtubules in cathepsin-inhibitor
treated, synchronized FRT cultures could help
determine whether mitotic catastrophe occurs
consequent to cathepsin inhibition.
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20
ACKNOWLEDGEMENTS
Thank you, ammi, for empowering me to realize my dreams. Your strength and kindness
has been beacons for me.
Thanks, little Dija, for inspiring me with your unbounded curiosity.
Thank you, Tanveer mamu, for believing in me.
I would like to thank Prof. Dr. Klaudia Brix for giving me the opportunity to work with
her, and the Brix group for welcoming me in its midst.
This thesis could not have been conceived without the guidance and encouragement of my
brilliant supervisor, Joanna Szumska.