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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.

3

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

4

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

6

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.