DRUG - INDUCED
MISLOCALIZATION OF RENAL PEROXISOMAL PROTEINS
D ISSERTATION
zur Erlangung des akademischen Grades des Doktors der Naturwissenschaften (Dr. rer. nat.)
vorgelegt von
L ISANNE L UKS
an der
Mathematisch-Naturwissenschaftliche Sektion Fachbereich Biologie
Tag der mündlichen Prüfung: 28. Juli 2017 1. Referent: Prof. Dr. Daniel R. Dietrich
2. Referent: Prof. Dr. Thomas Brunner 3. Referent: Prof. Dr. Marcel Leist
Konstanzer Online-Publikations-System (KOPS) URL: http://nbn-resolving.de/urn:nbn:de:bsz:352-0-421352
“We dance round in a ring and suppose, but the secret sits in the middle and knows.”
Robert Frost (1874 – 1963)
I 1.1 Journal articles ... V
1.2 Poster presentations ... V
1.3 Oral presentations ... VI
1.4 Funding ... VI
1.1 The renal proximal tubule ... 1
1.1.1 Anatomy and function ... 1
1.1.2 Proximal tubule cell models ... 3
1.2 Propiverine – a multi-functional drug ... 5
1.2.1 Pharmacological activity ... 5
1.2.2 Nonclinical findings in rats ... 7
1.3 D-amino acid metabolism ... 9
1.3.1 D-amino acids ... 9
1.3.2 D-amino acid oxidase (DAAO) ... 10
1.3.3 D-serine induced nephrotoxicity... 12
1.4 Peroxisomes ... 14
1.4.1 Organelle morphology ... 14
1.4.2 Organelle function ... 15
1.4.3 Import of peroxisomal proteins ... 16
II
3.1 Abstract ... 26
3.2 Introduction ... 26
3.3 Materials and Methods ... 29
3.4 Results ... 31
3.4.1 Recombinant DAAO properties are unaffected by propiverine ... 31
3.4.2 Propiverine induces loss of peroxisomal rDAAO localization and changes peroxisomal size... ... 32
3.4.3 Propiverine-induced mislocalization of peroxisomal catalase ... 34
3.4.4 Effects of propiverine on degradation pathways ... 34
3.5 Discussion ... 37
3.6 Supplementary material ... 40
4.1 Abstract ... 42
4.2 Introduction ... 42
4.3 Materials and Methods ... 44
4.4 Results ... 49
4.4.1 Impaired peroxisomal trafficking leads to cytosolic and enriched nuclear DAAO... 49
4.4.2 Interplay of multiple targeting signals and their influence on subcellular trafficking ... 51
4.4.3 The nuclear barrier is not rigid, allowing peroxisomal proteins to enter the nucleus depending on their molecular weight ... 52
4.5 Discussion ... 57
4.6 Supplementary material ... 59
5.1 Abstract ... 64
5.2 Introduction ... 65
5.3 Material and Methods ... 67
III
5.4 Results ... 70
5.4.1 Newly identified DAAO interaction partners in HEK293 cells ... 70
5.4.2 Mislocalization of several PTS1 matrix proteins after propiverine exposure ... 73
5.4.3 Propiverine interacts with several PTS1 proteins and enzymes involved in protein homeostasis ... 77
5.5 Discussion ... 80
5.6 Supplementary material ... 84
6.1 Abstract ... 92
6.2 Introduction... 92
6.3 Materials and Methods ... 95
6.4 Results ... 99
6.4.1 Culturing RPTEC/TERT1 cells in matrigel induces tubular morphology ... 99
6.4.2 3D cultured RPTEC/TERT1 cells form a highly polarized epithelium ... 101
6.4.3 Induction of transporter expression in tubular structures ... 103
6.4.4 Application as nephrotoxicity model ... 105
6.5 Discussion ... 107
6.6 Supplementary material ... 110
7.1 Nuclear mislocalization of proteins ... 113
7.1.1 Inclusion bodies in various tissues ... 113
7.1.2 Nuclear localization of peroxisomal proteins ... 114
7.2 Formation of protein accumulations in rat kidney ... 116
7.3 Similarity to peroxisomal disorders ... 118
7.4 Drug-specificity of protein accumulations ... 121
7.5 Establishment of a novel proximal tubule model system ... 124
7.6 Conclusion ... 125
IV
V Luks L., Sacchi S., Pollegioni L., Dietrich D.R. (2017). Novel insights into renal D-amino acid oxidase accumulation: propiverine changes DAAO localization and peroxisomal size in vivo. Arch Toxicol.
doi:10.1007/s00204-016-1685-z
Luks L.*, Maier M.Y.*, Sacchi S., Pollegioni L., Dietrich D.R. (2017). Understanding renal nuclear protein accumulation: an in vitro approach to explain an in vivo phenomenon. Arch Toxicol. doi:10.1007/s00204- 017-1970-5
Maier M.Y.*, Luks L.*, Wittmann, V., Dietrich D.R. (2017). A proteomic insight into in vitro interaction partners of D-amino acid oxidase and the drug propiverine. To be submitted to Chemico-Biological Interactions.
Luks L.*, Secker P.F.*, Schlichenmaier N., Dietrich D.R. (2017). RPTEC/TERT1 cells form highly differentiated tubules when cultured in a 3D matrix. To be submitted to ALTEX.
* shared first authors
Maier M.Y., Luks L., Dietrich D.R. (2017). Pharmaceutical-induced mislocalization and accumulation of peroxisomal proteins. 56th Annual Meeting of the Society of Toxicology (SOT), Baltimore, USA.
Schlichenmaier N., Luks L., Secker P.F., Dietrich D.R. (2017). Culturing RPTEC/TERT1 in 3D induces tubule formation and transporter expression. 83rd Annual Meeting of the German Society for Experimental and Clinical Pharmacology and Toxicology (DGPT), Heidelberg, Germany.
VI
Maier M.Y., Luks L., Dietrich D.R. (2016). Pharmaceutical-induced mislocalization and aggregation of PTS1 proteins. 4. Tagung der Experimentellen und Klinischen Pharmakologen und Toxikologen in Baden- Württemberg, Konstanz, Germany.
Luks L., Dietrich D.R. (2015). Unravelling the phenomenon of propiverine-induced DAAO accumulation in rat kidney: the role of proteasomal degradation. 54th Annual Meeting of the Society of Toxicology (SOT), San Diego, USA.
Maier M.Y., Luks L., Schlichenmaier N., Walz M., Dietrich D.R. (2015). Unravelling the phenomenon of propiverine-induced DAAO accumulation in rat kidney: manipulation of DAAO trafficking in vitro. 54th Annual Meeting of the Society of Toxicology (SOT), San Diego, USA.
Heussner A.H., Maier M.Y., Luks L., Secker P.F., Sacchi S., Pollegioni L. and Dietrich D.R. (2015). Unravelling the phenomenon of propiverine-induced DAAO accumulation in rat kidney: a direct approach. 54th Annual Meeting of the Society of Toxicology (SOT), San Diego, USA.
Microscopic investigation of propiverine-induced protein accumulation in rat kidney. (2016) APOGEPHA Arneimittel GmbH, Dresden, Germany.
Drug-induced renal accumulation of D-amino acid oxidase. (2016) University of Insubria, Department of Biotechnology and Life Sciences, Varese, Italy.
Research Training Group 1331 (RTG1331): Scholarship for PhD students funded by the Deutsche Forschungsgemeinschaft (DFG) (01/2014 – 04/2016).
Working group Human and Environmental Toxicology: Core funding, Prof. Dr. D.R. Dietrich, University of Konstanz (04/2016 – 06/2017).
VII Propiverine, a frequently prescribed drug for the treatment of overactive bladder and incontinence, provokes massive nuclear and cytosolic protein accumulations in rat kidney. These characteristic accumulations are a recurrent phenomenon arising in nonclinical safety studies and, since absent in mice and dogs, are believed to be rat-specific. However, the relevance for human risk assessment remains obscure, preventing i.a. drug approval of propiverine by the FDA. Ten years ago, one highly abundant constituent of the renal protein accumulation was identified as D-amino acid oxidase (DAAO), a peroxisomal enzyme. However, apart from this descriptive study, further mechanistical knowledge about this unexpected and anomalous nuclear localization of a peroxisomal protein is lacking.
The work presented in this thesis clarifies this adverse drug reaction by characterizing and elucidating the principal mechanisms underlying the formation of intranuclear and cytosolic protein accumulations. The composition of the in vivo accumulations was re-analyzed in detail revealing that not only DAAO but also several other peroxisomal proteins localize to nuclear and cytosolic accumulations. Strikingly, only proteins possessing a peroxisomal targeting signal 1 (PTS1) – but not PTS2 or peroxisomal membrane proteins – were found to be mislocalized or accumulated after propiverine treatment raising the question if propiverine interferes with proper peroxisomal trafficking of PTS1 proteins. Indeed, it was found that the drug interacts with several PTS1 proteins and peroxisomal chaperones crucial for correct folding and import of peroxisomal proteins, thus possibly causing impaired peroxisomal import. Subsequently, in order to study its abnormal nuclear localization, EYFP-tagged DAAO was modified and expressed in several cell lines to assess mechanisms involved in nuclear import of peroxisomal enzymes. Surprisingly, it was found that peroxisomal proteins of up to 135 kDa can passively diffuse into the nucleus, thereby contrasting textbook lines phrasing the ~40 kDa limit for passive diffusion through nuclear pores.
Furthermore, evidence is provided that peroxisomal proteins are degraded mainly via nuclear proteasomes under physiological conditions justifying the inherent nuclear presence of peroxisomal proteins. Besides its interaction with several PTS1 proteins, propiverine additionally interacts with two regulatory subunits of the 26S proteasome. Thus, it appears reasonable that propiverine hampers proteasomal degradation of PTS1 proteins converging in accumulation of the latter predominantly in nuclei. Using known proteasomal inhibitors, nuclear accumulation of DAAO could be induced in cell lines, however, not with propiverine assuming that standard cell lines do not fully reflect the in vivo morphology and function of a rat renal proximal tubule. Therefore, a highly-differentiated, functional proximal tubule cell model was established in the course of this thesis which better reflects in vivo-like characteristics e.g.
transporter expression. This 3D cell model of the proximal tubule might help to study phenomena like drug-induced protein accumulation which could not be mimicked in vitro so far.
In conclusion, this thesis reports that an interplay of distinct propiverine effects occurring in proximal tubules of rats results a priori in a mislocalization of several peroxisomal proteins and secondly causes accumulation of the latter possibly as consequence of an overwhelmed protein homeostasis.
VIII
Zur medikamentösen Behandlung von erhöhter Blasenaktivität und Inkontinenz wird häufig das Medikament Propiverin verschrieben, welches jedoch bei der Ratte zu massiven Proteinakkumulationen im Zellkern und Zytosol von Nierenzellen führt. Diese Akkumulationen scheinen ein wiederkehrendes Phänomen in präklinischen Studien zu sein und da sie weder in Mäusen noch Hunden zu beobachten sind, geht man von einem Ratten-spezifischen Effekt aus. Die FDA weigert sich dennoch bisher, das Medikament für den amerikanischen Markt zuzulassen, was vermutlich darauf zurück zu führen ist, dass das Risiko für den Menschen bisher ungeklärt ist. Vor 10 Jahren wurde ein peroxisomales Enzym, die D- Aminosäure Oxidase (DAAO), als Hauptbestandteil der renalen Proteinakkumulationen identifiziert.
Seitdem gibt es jedoch keine weiteren Studien, die diesen, für ein peroxisomales Protein außergewöhnlichen Fundort näher erklären.
Diese Thesis trägt maßgeblich dazu bei, diese Nebenwirkung näher zu charakterisieren und den zugrundeliegenden Mechanismus der intranukleären und zytosolischen Akkumulationen aufzuklären.
Hierzu wurde die Zusammensetzung der Proteinakkumulationen im Detail analysiert und es zeigte sich, dass nicht nur DAAO sondern zahlreiche weitere peroxisomale Enzyme mit einer peroxisomal targeting signal 1 (PTS1) Sequenz nach Propiverin-Gabe entweder mislokalisiert oder akkumuliert vorliegen. Dies ließ vermuten, dass Propiverin spezifisch den Transport von PTS1 Proteinen zu Peroxisomen stört.
Tatsächlich interagiert Propiverin direkt mit mehreren PTS1 Proteinen sowie peroxisomalen Chaperonen, die für die Faltung und den Transport von peroxisomalen Proteinen nötig sind. Um die nukleäre Proteinlokalisation zu klären, wurde EYFP-markiertes DAAO in verschiedenen Zelllinien überexprimiert und mutiert. Entgegen der langläufigen Meinung, dass nur Proteine bis ca. 40 kDa in den Kern diffundieren können, konnten wir zeigen, dass peroxisomale Proteine mit einer Größe bis zu 135 kDa passiv in den Kern diffundieren und dort auch unter physiologischen Bedingungen vorzufinden sind. Des Weiteren scheinen peroxisomale Proteine hauptsächlich in Kern-Proteasomen abgebaut zu werden, was einen Grund für deren nukleäre Lokalisation liefert. Da Propiverine an zwei regulatorische Untereinheiten des 26S Proteasoms bindet, ist es wahrscheinlich, dass Propiverin spezifisch den proteasomalen Abbau von PTS1 Proteinen stört, was letztlich zur Akkumulation führt. Tatsächlich führt ein bekannter Inhibitor des Proteasoms in verschiedenen Zelllinien zu nukleären DAAO-Akkumulationen, jedoch zeigt Propiverin in vitro keinen solchen Effekt. Dies legt die Vermutung nahe, dass Zelllinien in diesem Fall kein geeignetes Werkzeug sind, um komplexe in vivo Effekte nachzustellen. Aus diesem Grund wurde während dieser Thesis ein Tubulus-Modell etabliert, welches durch starke Zell-Differenzierung, Transporter-Expression und Funktionalität den Eigenschaften eines proximalen Tubulus ähnelt. Dieses 3D Modell des proximalen Tubulus kann dazu beitragen, dass bspw. Medikamenten-induzierte Proteinakkumulationen, die bisher in klassischen Zellkultur-Experimenten nicht nachzustellen waren, besser untersucht werden können.
Zusammenfassend zeigt diese Thesis, dass bei Propiverin mehrere Effekte im proximalen Tubulus von Ratten zusammenspielen: Zum einen führt Propiverin zur Mislokalisation einiger peroxisomaler Enzyme, zum anderen verhindert Propiverin wohl auch deren proteasomalen Abbau was schließlich in massiven Proteinakkumulationen im Kern und Zytosol in Rattennieren führt.
IX
3D three-dimensional
ABCD ATP-binding cassette sub-family D ACAA1 acetyl-coenzyme A acyltransferase 1
ACh acetylcholine
ACOX1 acyl-coenzyme A oxidase 1
ACTB β-actin
AGPS alkyldihydroxyacetone phosphate synthase ALPL alkaline phosphatase
ALS amyotrophic lateral sclerosis ANOVA analysis of variance
AQP1 aquaporin 1
ASP+ 4-(4-(dimethylamino)styryl)-N-methylpyridinium iodide ATP adenosine triphosphate
BCRP breast cancer resistance protein
BF bright field
Ca2+ calcium
CaM calmodulin
CAT catalase
CD circular dichroism
CoA coenzyme A
Co-IP co-immunoprecipitation
CRM1 chromosomal region maintenance 1 CTR1 copper transporter 1
CUBN cubilin
DAAO D-amino acid oxidase DnaJ chaperone DnaJ = HSP40
DTM docking/translocation machinery e.g. exempli gratia/ for example
EHHADH enoyl-CoA hydratase and 3-hydroxyacyl CoA dehydrogenase ER endoplasmatic reticulum
EYFP enhanced yellow fluorescent protein FAD flavin adenine dinucleotide
FDA food and drug administration f.i. for instance
GGT1 γ-glutamyltransferase 1 GLUT glucose transporter H2O2 hydrogen peroxide H2S hydrogen sulfide HAOX2 hydroxyacid oxidase 2 hDAAO human D-amino acid oxidase HEK human embryonic kidney
X
HPRT1 hypoxanthine phosphoribosyltransferase 1 HSC heat shock cognate
HSP heat shock protein i.a. inter alia/ among others i.e. id est/ that is
kDa kilo Dalton
Ki inhibitory constant
LAMP2 lysosomal associated membrane protein 2 LAP3 leucine aminopeptidase 3
LFQ label-free quantification
LY Lucifer yellow
MATE multidrug and toxin extrusion protein
mCh mCherry
MFE2 peroxisomal multifunctional enzyme type 2 mPTS membrane peroxisomal targeting signal MRP multidrug resistance protein
n.s. not significant
Na+ sodium
Na+/K+-ATPase sodium-potassium-ATPase nano-LC-ESI-
MS/MS
nano-scale liquid chromatography with electrospray ionization and tandem mass spectrometry
NES nuclear export signal
NH4+ ammonium
NLS nuclear localization signal NMDA N-methyl-D-aspartate
NOH61 ATP-dependent 61 kDa nucleolar RNA helicase NRK normal rat kidney
NSRI norepinephrine serotonin reuptake inhibitor NTS nuclear translocation signal
OAB overactive bladder OAT organic anion transporter OCT organic cation transporter
OCTN organic cation/carnitine transporter PBD peroxisome biogenesis disorder PEX peroxisomal biogenesis factor
PGP multidrug resistance protein/p-glycoprotein PKA protein kinase A
PKC protein kinase C
PMP peroxisomal membrane protein
PPAR peroxisome proliferator-activated receptor ppV preperoxisomal vesicle
PTS peroxisomal targeting signal rDAAO rat D-amino acid oxidase RFU relative fluorescence units
XI RNI relative nuclear intensity
ROS reactive oxygen species RPL13A 60S ribosomal protein L13a
RPN 26S proteasome regulatory subunit RPTEC renal proximal tubule epithelial cell
RT room temperature
SD standard deviation
SEM standard error of the mean SGLT sodium/glucose co-transporter SUMO small ubiquitin-like modifier TCP1 T-complex protein 1
TERT1 telomerase 1
WKPT Wistar-Kyoto kidney proximal tubule X-ALD X-linked adrenoleukodystrophy ZO-3 zona occludens protein 3 ZSS Zellweger syndrome spectrum α2u-globulin α2-microglobulin
XII
1 The kidney plays a fundamental role in maintaining the body’s salt, fluid and therefore blood pressure homeostasis. Its main function is to regulate the concentration of soluble substances by filtering the blood, reabsorbing desired solutes and excreting the remainder via the urine. To fulfill its functions, a kidney is built up of approximately one million nephrons, the functional units of the kidney (Bertram et al., 2011). The nephron is composed of a filtering component (glomerulus and Bowman’s capsule) and a tubular part (proximal tubule, loop of Henle, distal tubule). The initial segment filters soluble components of the blood into the tubular part of the nephron. The filtrate enters the proximal tubule and solutes like glucose, proteins and ions can be reabsorbed into highly polarized proximal tubule epithelial cells and further released into the surrounding peritubular capillaries. Indeed, the proximal tubule exerts the most prominent role in reabsorption compared to other nephron segments by accounting for the reabsorption of ~70% of all filtered load and most – if not all – filtered amino acids, solutes and low molecular weight proteins (Zhuo and Li, 2013). Furthermore, the proximal tubule is also the site of active secretion of xenobiotics making this part of the nephron particularly vulnerable for drug-induced nephrotoxicity.
The proximal tubule can be divided into the initial convoluted portion (pars convoluta) and a following straight portion (pars recta). On ultrastructural level, further division into S1, S2 and S3 segments is possible (Fig. 1). The S1 segment comprises the beginning and middle portion of the proximal tubule, the S2 segment includes the late portion of the convoluted tubule and the beginning of the straight tubule whereas the S3 segment comprises the remaining straight tubule portion (Zhuo and Li, 2013). On electron microscope resolution, individual characteristics of these segments can be found: The S1 segment contains cells with a wide brush border for solute reabsorption and multiple mitochondria and lysosomes possessing also the highest capacity for sodium, solute and protein transport of all renal tubular segments (Maunsbach, 1966). The following segment S2 bears shorter microvilli and less mitochondria but more lysosomes, whereas the S3 segment increases its brush border size and number of peroxisomes (Ohno, 1985; Zhuo and Li, 2013). Cells of the proximal tubule are highly polarized containing densely packed microvilli forming a brush border membrane on the apical side (Rhodin, 1958). The basolateral side is characterized by membrane invaginations and cells are strongly connected via tight junctions which are essential for proper polarization and active transport. For its indispensable role in reabsorption of solutes and ions, the proximal tubule expresses Na+/K+-ATPases at the basolateral membrane which are the main driving force for active transport across proximal tubules (Féraille and Doucet, 2001). Therefore, high
2
amounts of ATP are consumed by Na+/K+-ATPases maintaining low intracellular Na+ levels and a negative voltage which in turn provides the driving force for multiple transporters.
Besides the expression of a plethora of electrolyte, organic ion and glucose transporters in the proximal tubule, the transport and handling of proteins is also a predominant feature of the renal proximal tubule and will be mainly discussed here with focus on differences between rat and human. The glomerulus with its Bowman’s capsule provides a selective barrier for large molecules for entering the proximal tubule (Maack et al., 1979). Glomerular filtration decreases with molecular size, f.i. proteins up to 80 kDa can be filtered by the glomerulus and are heavily reabsorbed in the proximal tubule resulting in almost protein- free urine (Christensen et al., 2012). Proteins – but also vitamins, hormones, drugs and toxins – can enter the proximal tubule epithelial cells via receptor-mediated endocytosis at the apical side of the cells (Christensen et al., 2012). After binding to megalin and/or cubilin, megalin mediates the endocytosis of its ligand producing intracellular vesicles (Christensen et al., 2012). These vesicles fuse with lysosomes degrading their content and releasing amino acids or metabolized vitamins into the circulation (Christensen et al., 2012). Nevertheless, small traces of proteins are excreted via the urine including albumin and low molecular weight proteins (< 70 kDa) (Galaske et al., 1979). For example, albumin is partially filtered through the glomerulus (cplasma~45 g/L, cfiltrate~25 mg/L) and under physiological conditions, less than 1% of filtered albumin appears in the final urine (Gekle, 2005). Tubular malfunction, mainly caused by increased filtration after damage to the glomerular basement membrane, can result in proteinuria which is an important marker for kidney damage (Christensen and Gburek, 2004).
Fig. 1: Schematic of a nephron
A nephron consists of the glomerulus with Bowman’s capsule, the proximal tubule, the thin loop of Henle followed by the distal tubule (DCT). The proximal tubule can be divided into the initial convoluted portion (PCT) composed of the S1 and S2 segment. The following proximal straight tubule (PST) consists mainly of the S3 segment (grey). All parts of the proximal tubule as well as the glomerulus are localized in the cortex of the kidney. Adapted from (Manabe et al., 2014).
3 Importantly, not only increased filtered loads but also high amounts of poorly hydrolysable proteins can cause impaired protein degradation in the proximal tubule resulting in lysosomal overload (Maack et al., 1979; Miller and Palade, 1964). This phenomenon became of major interest when several chemicals were found to induce renal protein droplets and nephrotoxicity exclusively in male rats (Swenberg et al., 1989).
Subsequently, a low-molecular weight protein – α2-microglobulin (α2u-globulin) – was identified to be causal for this species- and sex-specific phenomenon (Alden, 1986). Low-molecular weight proteins are a heterogenous group of proteins which constitutes a small but biologically relevant fraction of total circulating proteins (Maack et al., 1979). This group of proteins includes enzymes (e.g. ribonucleases), immunoproteins (e.g. light chains of immunoglobulins, α/β2-microglobulins) and peptide hormones (e.g.
insulin) (Maack et al., 1979). Importantly, the kidney of male rats filters 50 mg of α2u-globulin per day, from which ~60% is reabsorbed and ~40% is excreted (Vandoren et al., 1983). This exceeds by far the amounts of α2u-globulin which are produced, filtered or excreted in female rats (Vandoren et al., 1983).
It was shown, that the toxicity-inducing chemicals reversely bind to α2u-globulin, stabilizing its structure and therefore reduce the lysosomal degradation of α2u-globulin in the proximal tubule (Swenberg et al., 1989). This results in an accumulation of α2u-globulin within lysosomes accompanied with cell shedding and restorative proliferation, finally leading to carcinogenesis (Swenberg, 1993; Swenberg et al., 1989).
This nephrotoxic mechanism helped to further characterize species-specific differences in renal protein handling since it was found that rats excrete ~90-fold more total protein than humans (Olson et al., 1990).
Of this excreted protein, the predominant fraction in rats consisted of low molecular weight proteins, whereas mainly high molecular weight proteins were found in humans (Olson et al., 1990).
As emphasized above, proximal tubule cells are highly differentiated and specialized cells with some species-specific characteristics e.g. high protein load in rat proximal tubule cells. For studying the physiology and pathophysiology of proximal tubule cells, several cell lines from rat and human origin are available.
The proximal tubule of rats is extensively studied in vivo, however cultivation of primary rat kidney cells remains difficult since they stop proliferating after some passages. Only a few rat kidney cell lines were established during the last decades, most prominently the NRK-52E and the WKPT cell line. A commonly used rat renal cell line is the NRK-52E (normal rat kidney) cell line derived from whole kidneys of Osborne- Mendel rats which was spontaneously immortalized (Duc-Nguyen et al., 1966) and later separated into epithelial-like (NRK-52E) and fibroblastic-like (NRK-49F) clones (de Larco and Todaro, 1978). NRK-52E cells show an epithelial morphology with tight junctions, respond to growth factors and exhibit well- characterized features for studying nephrotoxicity (Best et al., 1999). However, this cell line is not specific for one of the three segments within the proximal tubule (de Larco and Todaro, 1978; Duc-Nguyen et al., 1966). In contrast, the WKPT (Wistar-Kyoto kidney proximal tubule) cell line was derived from the S1 segment of the proximal tubule of Wistar-Kyoto rats and immortalized using SV40 large T-antigen (Woost
4
et al., 1996). The WKPT cell line is less popular but well characterized with regard to tight junctions, microvilli and active transport (Lee et al., 2007; Woost et al., 1996).
In contrast to rat renal cell lines, there are several human kidney cell lines available from which I will focus on the HEK293 and the RPTEC/TERT1 cell line. The HEK293 (human embryonic kidney) cell line was generated using kidney cells from a human fetus and was immortalized using adenovirus type 5 (Graham and Smiley, 1977). Due to their embryonic origin, HEK293 cells were found to resemble adrenal cells which have neuronal properties rather than reflecting a typical epithelial kidney cell (Shaw et al., 2002).
Nevertheless, HEK293 cells are widely used in cell biology since they grow rapidly in culture and are easy to transfect (Shaw et al., 2002). In contrast to HEK293 cells, the RPTEC/TERT1 (renal proximal tubule epithelial cell, telomerase 1) cell line was derived from human proximal tubule cells, immortalized by overexpression of human telomerase 1 and maintains multiple characteristics of renal proximal tubule cells e.g. cobblestone morphology, tight junctions, microvilli and active transport (Wieser et al., 2008).
Due to its maintenance of functionality and in vivo-like phenotype, this cell line is to date the most promising and widely used human proximal tubule model (Tiong et al., 2014; Wieser et al., 2008).
During the last decades, standard cell culture predominantly implied cultivation of cells on a plastic surface resulting in a monolayer of flattened cells. However, having the morphology and physiology of the renal proximal tubule in vivo in mind, it becomes obvious that these cultures differ dramatically from the in vivo architecture of a renal tubule composed of different cell types, extracellular matrix and interstitial fluids. Therefore, recent approaches use three-dimensional (3D) growth of cells in matrices like collagen or matrigel, allowing for improved morphology and functionality which increase the physiological relevance of cell models (Brien et al., 2002; Page et al., 2013).
5 In Europe and the United States, about 16% of adults suffer from overactive bladder (OAB) and urinary incontinence with symptoms such as frequency, urgency, urge incontinence and nocturia (Ganz et al., 2010; Milsom et al., 2001). Although not life-threatening, OAB is an economically costly condition and is highly related to a decrease of patient’s life quality (Bartoli et al., 2010; Ganz et al., 2010). Whereas previously mainly elderly people were affected by OAB, increasing incidences of overweight and obesity contribute significantly to the risk for urinary incontinence also in younger people (Subak et al., 2009).
The International Continence Society classifies OAB as a syndrome without precise cause e.g. organ abnormalities (Ouslander, 2004). OAB symptoms result from an excessive stimulation of bladder contraction or reduced relaxation of the detrusor muscle during bladder filling, either by neurogenic or idiopathic overactivity (Ouslander, 2004). Acetylcholine, an agonist of muscarinic receptors in the detrusor muscle, is the main contractive transmitter in the bladder (Ouslander, 2004). In the detrusor muscle, muscarinic receptors of the M2 and M3 subtype are predominantly expressed, both are coupled to G proteins, but the M3 receptor is most important for detrusor contraction (Abrams and Andersson, 2007). In general, muscarinic M3 receptors induce several signaling pathways e.g. activation of phospholipase C with subsequent formation of inositol triphosphate and release of Ca2+ from intracellular stores, as well as activation of other phospholipases (phospholipase D and A2) and kinases (protein kinase C, phosphatidylinositol-3-kinase, tyrosine kinase, mitogen-activated kinase and Rho kinase) and opening of ion channels (Frazier et al., 2008).
Regarding bladder contraction, it could be shown that binding of acetylcholine to M3 receptors mainly triggers activation of kinases (protein kinase C, Rho kinase) and elevation of intracellular Ca2+
concentrations (Fig. 2), whereas phospholipases show little contribution (Frazier et al., 2008; Schneider et al., 2004; Takahashi et al., 2004). Intracellular Ca2+ concentration increase can be mediated either via mobilization from intracellular stores or from extracellular influx of Ca2+ mainly through voltage- dependent L-type Ca2+ channels and is – similar to all other types of smooth muscle – required for bladder contraction via calmodulin-mediated activation of myosin light chain (MLC) kinase (Fig. 2) (Schneider et al., 2004; Takahashi et al., 2004). In the bladder, coupling of muscarinic receptor activation to opening of L-type Ca2+ channels is thought to be mediated via phosphorylation of the Ca2+ channel subunits by protein kinase C (Fig. 2) (Lin et al., 1998; Liu and Lin-Shiau, 2000). First-line pharmacological therapy for OAB are anticholinergic drugs since they effectively suppress premature detrusor contraction (Hesch, 2007).
Propiverine – sold under the trade names Mictonorm® and Detrunorm® – is one of the antimuscarinics which is recommended by the Committee on Pharmacological Treatment as medication for OAB (Madersbacher and Mürtz, 2001). Today, propiverine is marketed in 23 countries all over Europe and Asia (being the most frequently prescribed anticholinergic drug in Japan), but has no approval in the United
6
States and Canada (Ramsay and Bolduc, 2017). Although propiverine is a non-selective muscarinic receptor antagonist (Wuest et al., 2006), it shows same efficiency in treatment of OAB than other pharmaceuticals in clinical use like oxybutynin, but provides only mild side-effects e.g. dry mouth (Abrams and Andersson, 2007). Besides its antimuscarinic effect, propiverine was shown to exert several spasmolytic effects such as calmodulin binding (Matsushima et al., 1997) and L-type Ca2+ channel antagonism (Uchida et al., 2007). Furthermore, propiverine inhibits protein kinase C, thereby enhancing the blockage of L-type Ca2+ channels (Möritz et al., 2005). Propiverine differs even more from other OAB drugs since it is also a potent antagonist of α1-adrenoceptors (Wuest et al., 2011) and an inhibitor of noradrenalin reuptake (Nishijima et al., 2016). This broad mode of action most likely amplifies the clinical effect of propiverine on the bladder, nevertheless, the exact mechanism of this interplay remains to be determined (Frazier et al., 2008).
Upon oral administration, propiverine is rapidly absorbed and undergoes extensive metabolism to its main hepatic metabolite propiverine-N-oxide (Fig. 3) (Haustein and Huller, 1988). The N-oxide metabolite shows higher plasma and urine concentrations than the parent compound (Haustein and Huller, 1988) but is less efficient in binding to muscarinic receptors (Wuest et al., 2006), L-type Ca2+ channels (Uchida
Fig. 2: Schematic of acetylcholine(ACh)-induced muscle contraction in the bladder and interaction sites of propiverine
Main pathways for M3 receptor-mediated contraction are the inhibition of myosin light chain (MLC) phosphatase through activation of Rho kinase and protein kinase C as well as activation of MLC kinase via calmodulin/Ca2+-complexes. Ca2+ is necessary to activate protein kinase C, the latter opens L-type Ca2+
channels by phosphorylation (P). MLC kinase phosphorylates MLC thereby inducing contraction.
Interaction sites of propiverine are indicated by red asterisks.
7 et al., 2007), protein kinase C (Möritz et al., 2005) and α1-adrenoceptors (Wuest et al., 2011). Overall, propiverine has several active metabolites that differ in their quantitative and qualitative characteristics and contribute with variant degree to the desired pharmacological effect, the inhibition of premature bladder contraction (Michel and Hegde, 2006).
Propiverine was developed by Apogepha Arzneimittel GmbH, Dresden, Germany and first registered in the German Democratic Republic in 1981 (Dietrich et al., 2008). When tested for drug approval and marketing authorization in Japan, propiverine revealed unexpected side effects in nonclinical studies with rats. Propiverine resulted in massive eosinophilic inclusions in rat kidney cortex after 13- and 52-week oral administration (Nakano et al., 1989; Yamashita et al., 1990). Interestingly, the drug-induced inclusions were exclusively found in the S2 and S3 segments of proximal tubule cells but did not occur elsewhere in the kidney or in the body (Dietrich et al., 2008). Within the proximal tubule epithelial cells, large, spherical inclusions were found both in the cytosol and nuclei (Dietrich et al., 2008; Nakano et al., 1989; Yamashita et al., 1990). The latter resulted in distinct displacement of the chromatin (Dietrich et al., 2008). However, at that time, the composition of the inclusions was unknown, only a homogeneous eosinophilic staining (Nakano et al., 1989; Yamashita et al., 1989b) allowed to assume a proteinous content. Indeed, using laser-caption microdissection and mass spectrometry, the renal inclusions were identified as protein accumulations consisting of predominantly one protein: D-amino acid oxidase (DAAO) (Dietrich et al., 2008). Under physiological conditions, DAAO is located in peroxisomes (Sacchi et al., 2012) and – to the best of my knowledge – was never found before to be localized or even accumulated in the nucleus.
Besides renal DAAO accumulations, rats treated with propiverine for 13 or more weeks presented alterations in serum and urine analyses. Rats showed reduced body weights, increased urine volume with increased excretion of electrolytes, decreased serum triglyceride and cholesterol levels and increased γ- glutamyltransferase levels (Nakano et al., 1989; Yamashita et al., 1990) indicating impaired kidney and
Fig. 3: Chemical structures of propiverine and its main metabolite propiverine-N-oxide
Chemical structures of the benzylic acid derivative propiverine (1-methylpiperidin-4-yl-2,2-diphenyl-2- propoxyacetate) and its main metabolite propiverine-N-oxide which results from oxidation of the tertiary nitrogen in the piperidinyl moiety by monooxygenases (May et al., 2008). Modified from (Wuest et al., 2005).
8
liver function. Although being highly abundant, renal protein accumulations did not trigger histopathological lesions or elevated tumor rates in the kidney (Dietrich et al., 2008). However, autopsy revealed anomalies of the liver e.g. liver hypertrophy, midzonal lipid droplets and hyperplasia of smooth endoplasmatic reticulum in hepatocytes (Nakano et al., 1989; Yamashita et al., 1990). Importantly, after a 4-5-week recovery period, all above-mentioned side effects massively decreased or disappeared, suggesting reversible side-effects of propiverine (Kohda et al., 1989; Nakano et al., 1989; Yamashita et al., 1990). Since liver effects – but not renal accumulations – were also found in a 13-week and 1-year study with beagle dogs (Kohda et al., 1989; Yamashita et al., 1989a), this raises the question whether propiverine affects the liver in several species but provides a rat-specific effect with regard to renal protein accumulations.
Interestingly, there is evidence that not only propiverine, but at least two other pharmaceuticals provoke cytosolic and intranuclear DAAO accumulations in rat kidney (Gopinath and Mowat, 2014; Radi et al., 2013). However, only one company disclosed their nonclinical findings involving a norepinephrine- serotonin reuptake inhibitor (NSRI) inducing DAAO accumulations in rat kidneys after 4-week exposure (Radi et al., 2013). Moreover, renal DAAO accumulations were sporadically observed in wild-type Wistar Hannover rats with an incidence rate of <5% (Shimoyama et al., 2014). The published articles are predominantly descriptive and identified the presence of DAAO within accumulations using DAAO- specific antibodies without analyzing the protein content in more detail. Furthermore, none of them provides evidence for the underlying mechanism of drug-induced or spontaneous DAAO accumulation in rat kidney. So far, to the best of my knowledge, no reports about similar drug-induced protein accumulations in humans are released to the public.
9 Today it is widely accepted that not only L- but also the non-proteinogenic D-amino acids play important physiological and pathophysiological roles in pro- and eukaryotes (Pollegioni et al., 2007). Recent progress in analytical methods revealed that free D-amino acids are predominantly found in the brain and in traces in blood and urine in humans, whereas rats and mice show also significant D-amino acids levels in kidney, liver and testis (Hamase et al., 2002). D-amino acids are taken up with the diet (processed food and drinks contain high levels of D-amino acids) or arise through spontaneous racemization of L-amino acids (Martínez-Rodríguez et al., 2010). Recently, the microbe-rich intestinal flora gained attention as source for D-amino acids since peptidoglycan, the predominant component of bacterial cell walls, incorporates D-alanine and D-glutamic acid (Radkov and Moe, 2014). Only two different amino acid racemases are reported in mammals; serine and aspartate racemase, both are found exclusively in the brain (Ohide et al., 2011).
In general, D-amino acids are related to signaling, protective or toxic features of the cell (Martínez- Rodríguez et al., 2010) and high levels are a main characteristics of aging (D’Aniello et al., 1993) as well as of amyotrophic lateral sclerosis (ALS) (Sasabe et al., 2007) and Alzheimer’s disease (D’Aniello et al., 1992) and renal failure (Hamase et al., 2002; Kimura et al., 2016). Among the most studied and most abundant D-amino acids in the brain, D-aspartate and D-serine should be mentioned which are involved in binding to the N-methyl-D-aspartate (NMDA)-type glutamate receptor (Martínez-Rodríguez et al., 2010).
In the kidney, 99% of all amino acids are filtered by the glomerulus and reabsorbed mainly by the proximal tubule (Zelikovic and Chesney, 1989). In general, amino acid transport systems accept groups of amino acids e.g. basic or neutral amino acids (Bröer, 2008). Transport into proximal tubule cells is predominantly mediated via apical Na+-cotransporters, followed by Na+-independent facilitated diffusion across the basolateral membrane (Rabito and Karish, 1983; Verrey et al., 2009). There is contradictory information about stereoselectivity of amino acid transport systems, some seem to be non-selective, whereas others reabsorb D-amino acids more slowly than the respective L-isoforms (Bröer, 2008; Silbernagl and Völkl, 1977). One study addressed D-serine uptake into the proximal tubule of rats, concluding that D-serine is only reabsorbed in the S3 segment by a high-capacity/low-affinity transporter at similar rates than L- serine (Silbernagl et al., 1999).
10
D-amino acid oxidase (DAAO; EC 1.4.3.3) was discovered in pig kidney more than 80 years ago (Krebs, 1935) and since then it has been extensively studied as a prototype of flavin-dependent oxidases. DAAO catalyzes the oxidative deamination of D-amino acids into its α-keto acids thereby generating hydrogen peroxide (H2O2) (Fig. 4).
DAAO is expressed in numerous eukaryotic organisms including yeasts, insects and mammals like mice, rats, dogs and humans (Pollegioni et al., 2007). In mammals, DAAO is expressed mainly in three organs:
kidney, liver and brain (descending order of expression level) (Khoronenkova and Tishkov, 2009). In mice, DAAO expression is restricted to the kidney and the brain, lacking expression in the liver (Konno et al., 1997). Within the kidney, DAAO is expressed exclusively in the proximal tubules of the renal cortex where expression is highest in the pars recta (S2/S3 segment) (Angermüller and Fahimi, 1988; Usuda et al., 1986a).
Except for species- and tissue-specific expression patterns, there are several structural differences of rat and human DAAO which are of special interest regarding the supposed rat-specific renal DAAO accumulations after propiverine treatment. Rat DAAO (rDAAO) shares 80% sequence homology with human DAAO (hDAAO) (Konno, 1998); nonetheless rDAAO differs significantly from the human counterpart: (1) rDAAO is active as a monomer (Frattini et al., 2011) while hDAAO is a stable dimer (Molla et al., 2006), (2) rDAAO shows lower kinetic efficiency and a different substrate specificity as compared to the human counterpart (Frattini et al., 2011) and (3) rDAAO consists of 346 amino (38.8 kDa), whereas hDAAO consists of 347 amino acid residues (39.5 kDa) (Konno, 1998).
In eukaryotic cells, DAAO is synthesized at free ribosomes, released into the cytosol where it is immediately bound by chaperones assisting in proper protein folding (Dias et al., 2016). All known DAAOs
Fig. 4: Schematic reaction catalyzed by DAAO
DAAO catalyzes the deamination of D-amino acids into its imino acids, thereby reducing the cofactor flavin adenine dinucleotide (FAD) to FADH2. The imino acid reacts spontaneously with water to the corresponding α-keto acid and ammonium (NH4+). FADH2 is reoxidized, thereby releasing hydrogen peroxide (H2O2) (Pollegioni et al., 2007).
11 contain a C-terminal peroxisomal targeting signal 1 (PTS1) allowing for guided transport into peroxisomes (Sacchi et al., 2012). Due to the H2O2-producing activity of DAAO, this subcellular compartmentalization protects the cell from reactive oxygen species (ROS) by providing immediate peroxisomal degradation of H2O2 via catalase (Usuda et al., 1986a). Like most peroxisomal proteins, hDAAO is a long-lived protein with an estimated half-life of ~60 h (Cappelletti et al., 2013). In glioblastoma cells it is suggested, that peroxisomal hDAAO is degraded via the lysosomal pathway, whereas cytosolic hDAAO can be ubiquitinylated and degraded via the proteasome system (Cappelletti et al., 2013).
Besides DAAO, there are several enzymes acting on D-amino acid metabolism e.g. amino acid-specific oxidases such as D-aspartate oxidase, D-alanine transaminase or serine racemase (Martínez-Rodríguez et al., 2010).
As described above, DAAO degrades D-amino acids with strict stereospecificity and therefore specifically regulates D-amino acids levels in the body concomitantly providing new building blocks for protein synthesis by producing α-keto acids (Pollegioni et al., 2007). In all physiological but also pathophysiological processes DAAO is involved in, the underlying mechanism is the regulation of D-amino acid levels indicated by inversely correlated presence of DAAO and D-amino acids within one tissue (Sacchi et al., 2012). After conversion of D-amino acids to α-keto acids, these intermediates are further converted to L-amino acids by transaminases and used as building blocks for protein synthesis (Hasegawa et al., 2004). It was shown, that ~30% of an administered dose of D-leucine is converted to the L- enantiomer through the α-ketoisocaproic acid intermediate by renal DAAO (Hasegawa et al., 2004). Mice or rats lacking DAAO expression exhibit highly elevated D-amino acids levels, also excreting high amounts via the urine indicating that the kidney is deeply involved in D-amino acids metabolism (Konno et al., 1989; Yamanaka et al., 2012).
Research on DAAO focuses primarily on the brain, nevertheless the physiology of DAAO within other sites of expression e.g. the kidney lately became more and more important revealing – in contrast to the brain – protective roles of DAAO within the kidney. Recently, Kimura and coworkers identified elevated plasma levels of D-amino acids as novel biomarkers for chronic kidney disease, age and diabetes mellitus in humans (Kimura et al., 2016). They hypothesized that acute and chronic kidney injury decreases DAAO activity in the proximal tubule leading to an increase in D-amino acids in the plasma which then worsens the kidney function (Kimura et al., 2016). This is further supported by the fact that mice subjected to ischemia-reperfusion injury exhibited a sharp increase in serum and urine D-serine levels concomitant with loss of DAAO-positive proximal tubules (Sasabe et al., 2014). Taken together, the kidney and renal DAAO may play an important role in maintaining the levels of D-amino acids in the blood by regulating reabsorption/excretion and metabolization, respectively.
DAAO was also found to be involved in hydrogen sulfide (H2S) production in cerebellum and kidney of mice (Shibuya et al., 2013). DAAO (together with the mitochondrial enzyme 3-mercaptopyruvate sulfurtransferase) catalyzes H2S production from D-cysteine (Shibuya et al., 2013). H2S can enhance the
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activity of NMDA receptors, relaxes vascular smooth muscles by activating K+ channels and regulates the release of insulin (Shibuya et al., 2013). Furthermore, H2S has a cytoprotective function by recovering glutathione levels, scavenging ROS (Kimura et al., 2010) and decreasing intracellular Ca2+ concentrations by inhibiting L-type Ca2+ channels (Zhang et al., 2012). As the H2S producing enzymes e.g. DAAO are located in the renal cortex, this part – but not the medulla – was protected by administration of D-cysteine after ischemia-reperfusion (Shibuya et al., 2013).
As mentioned before, research on DAAO mainly focuses on the brain where DAAO metabolizes D-serine thereby regulating the activity of NMDA receptors (Verrall et al., 2010). As a result, DAAO has a pivotal role in motoneuron degeneration and is involved in Parkinson’s disease (Kawazoe et al., 2007), schizophrenia (Chumakov et al., 2002) and familial ALS (Mitchell et al., 2010). The main pathological hallmark of Parkinson’s disease is the loss of dopamine-producing neurons resulting in depletion of dopamine levels which manifest in typical symptoms like tremor, rigor and akinesia (Lotharius and Brundin, 2002). D-DOPA, the stereoisomer of L-DOPA, is commonly used in the treatment of Parkinson’s disease and can be metabolized by DAAO (Kawazoe et al., 2007). DAAO is involved in the conversion of
D-DOPA into L-DOPA which in turn can be converted into dopamine (Kawazoe et al., 2007). D-DOPA oxidation velocity by DAAO is significantly faster compared to other DAAO substrates e.g. D-serine, indicating that D-DOPA is a preferred substrate for human DAAO (Kawazoe et al., 2007).
The possibility that DAAO is also pathophysiologically involved in schizophrenia advanced by the finding that both DAAO expression and activity are increased in patients with this disorder (Verrall et al., 2010).
Concomitantly, schizophrenic patients show decreased D-serine levels and NMDA receptor hypofunction (Verrall et al., 2010). Interestingly, long before DAAO was linked to schizophrenia, the two antipsychotics chlorpromazine and risperidone (primarily inhibiting dopamine receptors) were effectively used to treat schizophrenia and were both shown to be DAAO inhibitors (Verrall et al., 2010). However, so far no specific DAAO inhibitor reached the market, likely because DAAO inhibition alone cannot achieve the desired efficacy and a sharp increase in brain D-serine would rise the potential for oxidative damage (Verrall et al., 2010).
D-amino acid induced nephrotoxicity is a species-specific phenomenon where DAAO reveals its dangerous side: the production of H2O2 and the consequences thereof. Several researchers found that intraperitoneal injection of D-serine results in nephrotoxicity in different rat strains but not in mice, rabbits, dogs, hamsters, guinea pigs or gerbils (Maekawa et al., 2005). Within hours after D-serine injection, cells of the S3 segment of the proximal tubule undergo necrosis accompanied by glucosuria and proteinuria (Ganote et al., 1974; Krug et al., 2007). Interestingly, neither D-alanine nor D-proline are nephrotoxic (Kaltenbach et al., 1979). However, presence of DAAO was shown to be causal for the observed toxicity since LEA/SENDAI rats, lacking DAAO activity, are resistant to toxicity (Maekawa et al., 2005). In presence of its substrate D-serine, DAAO activity results in high levels of H2O2 which in turn is responsible for the severe toxicity in proximal tubules (Maekawa et al., 2005). Nephrotoxicity could be
13 prevented by co-treatment with a DAAO inhibitor (Williams and Lock, 2005) or supplementation with reduced glutathione (Krug et al., 2007). It is hypothesized that D-serine which is not reabsorbed in S1 or S2 segments of the kidney, is highly concentrated due to water reabsorption until it reaches the S3 segment (Krug et al., 2007). In the S3 segment – where DAAO is also highest expressed – D-serine is reabsorbed and metabolized yielding local toxic H2O2 concentrations (Krug et al., 2007). Still, this does not explain the species-specific component of D-serine induced nephrotoxicity. All of the above mentioned species have comparable DAAO activity levels, therefore it is hypothesized that reabsorption of D-amino acids differs within the species (Maekawa et al., 2005). Indeed, rats excrete very little amounts of D-amino acids compared to humans and dogs while having comparable serum concentrations (Huang et al., 1998) indicating that D-amino acids are heavily reabsorbed and metabolized in rats. In addition, rats have a higher capacity to use D-amino acids and α-keto acids for protein synthesis compared to other animals (Maekawa et al., 2005). Nevertheless, there is one report about D-propargylglycine induced nephrotoxicity which is also dependent on DAAO activity but which is not restricted to rats but occurs also in mice (Konno et al., 2000). Therefore, a species-specific handling of D-amino acids in the kidney with better reabsorption and metabolization rates in rats than in humans appears likely to cause D-serine induced nephrotoxicity in rats (Huang et al., 1998; Krug et al., 2007; Maekawa et al., 2005).
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Peroxisomes perform a large variety of metabolic functions and are present in virtually all eukaryotic cells (Lodhi and Semenkovich, 2014). Besides their multiple functional capacities, these organelles vary in size, number and morphology between organisms and even cell types or developmental stages of the same organism (Baker and Paudyal, 2014). Peroxisomes are small, single membrane-enclosed organelles containing a granular matrix and occasionally possessing a crystalline core (Titorenko and Rachubinski, 2001). The assembly, function and growth of peroxisomes is regulated by proteins called peroxins (PEX) (Schrader and Fahimi, 2008). So far, about 20 PEX proteins have been discovered in mammalians which are required for biogenesis and maintenance of functional peroxisomes (Schrader and Fahimi, 2008).
Mammalian cells contain several hundred peroxisomes (Lazarow and Fujiki, 1985) and besides the liver, peroxisomes are most abundant in the kidney (Islinger et al., 2010). Consistent with the expression of DAAO which is highest in the S3 segment of the proximal tubule, peroxisomes are most abundant in this segment of the pars convoluta (Fig. 5, left) (Zhuo and Li, 2013).
Fig. 5: Peroxisomes in proximal tubule epithelial cells of rats and HepG2 cells
Left: Electron microscopy of proximal tubule epithelial cells of rats. Peroxisomes (P) are most abundant in the S3 segment. L = lysosomes. Adapted from (Zhuo and Li, 2013).
Top: Different cells in the same culture show different peroxisome morphology. Immunofluorescence microscopy of HepG2 cells stained for PMP70 (A) or acyl-CoA oxidase (C) and 3D reconstructions of electron microscopy (B + D).
A + B: Spherical peroxisomes, some arranged like beads on a string (arrow) but showing no interconnection. C + D:
Elongated, tubular peroxisomes. Adapted from (Grabenbauer et al., 2000).
15 Congruent with their function, peroxisomes are markedly dynamic organelles. Peroxisomes are 0.1 – 1 µm in diameter and mostly spherical but can change into round or elongated shapes in certain cell types or environments (Fig. 5, top) (Smith and Aitchison, 2013). Whereas liver and kidney contain large and round peroxisomes, other tissues express so called microperoxisomes (0.1 – 0.4 µm) which show overall the same morphology but are much smaller in size (Antonenkov and Hiltunen, 2012).
Early electron microscopy studies of the kidney identified small cytoplasmic, single-membrane
“microbodies” (Rhodin, 1958), later termed peroxisomes referring to their hydrogen peroxide metabolism, the first identified peroxisomal function (De Duve and Baudhuin, 1966). Today, it is known that peroxisomes catalyze a number of essential metabolic functions including fatty acid β-oxidation, ether phospholipid biosynthesis, glyoxylate detoxification and degradation of purines, polyamines, D- amino acids and hydrogen peroxide (Visser et al., 2007). Metabolic function requires constant flow of metabolites across the peroxisomal membrane. Contrary to enzymes of other organelles, peroxisomal enzymes do not increase their activity after disruption of the organelle membrane, indicating that the peroxisomal membrane is open to solutes and/or contains pore-forming proteins (Antonenkov and Hiltunen, 2012). However, some studies indicated that the peroxisomal membrane is impermeable for NAD+ and contains ATP transporters (Antonenkov and Hiltunen, 2006). Today it is widely accepted that the peroxisomal membrane uses pore-forming channels (PXMP2/PMP22) (Rokka et al., 2009) for transfer of solutes <300 Da e.g. substrates of peroxisomal oxidases (urate, glycolate and D-amino acids) (Antonenkov and Hiltunen, 2006) but restricts the diffusion of bulky molecules like ATP, NAD+, FAD and CoA for which the membrane contains transporters (Antonenkov and Hiltunen, 2012). So far, two members of the mitochondrial solute carriers have been identified in peroxisomes: an ATP transporter called peroxisomal membrane protein 34 (PMP34, SCL25A17) (Visser et al., 2002) and a Ca2+-dependent transporter (Weber et al., 1997). Whereas peroxisomal ATP transporters are assumed to efficiently transport NAD+, FAD and ATP, the transport of CoA could so far only been shown in plants assuming that free CoA accumulates within mammalian peroxisomes (Antonenkov and Hiltunen, 2012). The uptake of substrates for β-oxidation is mediated via members of the ATP-binding cassette (ABC) superfamily D (Kemp et al., 2011). In peroxisomal membranes of mammalian cells, three members of the ABCD family are expressed: ABCD1 (adrenoleukodystrophy protein, ALDP), ABCD2 (adrenoleukodystrophy-related protein ALDR) and ABCD3 (70 kDa peroxisomal membrane protein, PMP70) (Baker et al., 2015). All three ABC transporters use ATP to import CoA esters of fatty acids with distinct but overlapping specificities for the different CoA esters (Baker et al., 2015). Until now, it remains to be determined whether or how the large CoA moiety (770 Da) is transported or cleaved off during transport (Baker et al., 2015).
β-oxidation, the process by which fatty acids are degraded from their C-terminal end, is the best studied metabolic function of peroxisomes and involves the majority of peroxisomal enzymes (Visser et al., 2007).
Peroxisomal β-oxidation of very long- and long-chain fatty acids is limited to a few cycles of oxidation resulting in medium-chain fatty acids and acetyl-CoA. To export these products, peroxisomes need to
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separate the corresponding moieties from CoA enabling the exit into the cytosol. Several peroxisomal thioesterases catalyze the hydrolysis of CoA derivates to CoA and free fatty acids, bile acids etc. The latter can freely pass the peroxisomal membrane through channels and are further metabolized in other cellular compartments e.g. mitochondria. Therefore, peroxisomes provide cells with metabolic precursors which can be further metabolized in other cellular compartments. (Antonenkov and Hiltunen, 2012)
As mentioned before, the first identified function of peroxisomes – accounting for its name – was the metabolization of H2O2 (De Duve and Baudhuin, 1966). In peroxisomes of mammals, about 10 ROS- generating enzymes were found, all of them are oxidases reducing O2 and yielding H2O2 (Schrader and Fahimi, 2006). The high peroxisomal consumption of O2, production of ROS – particular of H2O2 – and the high abundance of several ROS-metabolizing enzymes support the key roles of peroxisomes: the production and scavenging of ROS (Schrader and Fahimi, 2006). It is estimated that ~35% of all H2O2
formed in rat liver derives from peroxisomal oxidases (Boveris et al., 1972). To degrade ROS, peroxisomes contain several antioxidant enzymes including the catalase, superoxide dismutases, glutathione peroxidase and peroxiredoxin 1 (Schrader and Fahimi, 2006). Catalase – the classic marker protein for peroxisomes – is one of the key players in protection against ROS in peroxisomes, being highly efficient in removing H2O2 yielding H2O and O2 (Schrader and Fahimi, 2006).
All peroxisomal membrane and matrix proteins are synthesized in the cytosol and are post-translationally imported into the organelle (Lazarow and Fujiki, 1985). Folding of the newly synthesized polypeptide chain is catalyzed by several cytosolic chaperones and probably starts as soon as the first N-terminal amino acids emerge from the ribosome (Titorenko and Rachubinski, 2001). The cytosolic chaperone complex consisting of T-complex protein ring complex (TriC), heat shock protein 40 (HSP40) and HSP70 functions co-translationally, whereas the heat shock cognate 70 (HSC70) acts post-translationally (Titorenko and Rachubinski, 2001). While proteins of the ER and mitochondria can cross their respective membranes only in an unfolded, monomeric state, peroxisomal matrix proteins can be imported when completely folded and in oligomeric states (Titorenko and Rachubinski, 2001).
To be sorted into the peroxisomal matrix, newly synthesized proteins contain a peroxisomal targeting signal (PTS). So far, two types of PTS are known and well-characterized: the PTS1 and PTS2 sequence. The PTS1 is most prevalent and consists of a C-terminal three-amino acid sequence (S-K-L) (Subramani, 1993).
The PTS2 is very rare – only three peroxisomal matrix proteins are known to possess a PTS2 (Schlüter et al., 2009) – and presents as nine-amino acid sequence at the N-terminus (Lazarow, 2006; Legakis and Terlecky, 2001). In contrast to the PTS1 sequence which is not processed after import into the peroxisome, the PTS2 is cleaved off within the peroxisomal matrix (Kurochkin et al., 2007). Sorting of
17 PTS1 proteins requires the peroxisomal biogenesis factor 5 (PEX5), the cytosolic receptor for PTS1 proteins. There are two known isoforms of PEX5, a short PEX5S form acting as PTS1 receptor and a longer PEX5L isoform which is required for the import of PTS2 proteins into peroxisomes (Braverman et al., 1998). PEX5S recognizes the PTS1 of newly synthesized, cytosolic proteins and docks them to the surface of the peroxisomal membrane to initiate import (Fig. 6) (Titorenko and Rachubinski, 2001). PEX7 binds to PTS2 sequences and – together with PEX5L – mediates docking at the docking/translocation machinery (DTM) (Legakis and Terlecky, 2001). After interaction with PEX14, both PTS1- and PTS2-receptor-cargo complexes are transferred to a membrane-associated complex consisting of the RING finger proteins PEX2, PEX10 and PEX12 (Fig. 6) (Rodrigues et al., 2015). Then, the receptor-cargo complexes are inserted into the DTM with concomitant translocation of the cargo proteins across the peroxisomal membrane (Rodrigues et al., 2015). PEX5 remains within the DTM, developing a transmembrane topology facing the peroxisomal matrix (Rodrigues et al., 2015). PEX7 remains bound to PEX5 during most steps of the PTS2- mediated protein import (Rodrigues et al., 2016). Interestingly, the transport process needs no external driving force but results exclusively from strong protein-protein interactions of PEX receptors and PEX proteins of the DTM (Rodrigues et al., 2015). To recycle the receptors, PEX5 is monoubiquitinated and
Fig. 6: Import of matrix proteins into peroxisomes in mammalian cells
The peroxisomal targeting signals PTS1 and PTS2 are recognized by the cytosolic receptors PEX5S and PEX7, respectively. The long isoform of PEX5 (PEX5L) forms a complex with the PEX7-cargo-complex. Both PEX5S and PEX7L interact with PEX14 resulting in transfer of the protein-receptor-complexes to a membrane-associated complex consisting of PEX2, PEX10, PEX12 and PEX13. The protein-receptor- complexes are translocated into the peroxisomal matrix where the protein is released and the receptors are recycled into the cytosol by PEX2, PEX1 and PEX6 (dotted lines). (Titorenko and Rachubinski, 2001)
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extracted back into the cytosol in an ATP-dependent manner by a complex consisting of the two ATPases PEX1 and PEX6 (Fig. 6) (Rodrigues et al., 2015).
Interestingly, some peroxisomal matrix proteins have been described to lack any detectable PTS-motifs but are nonetheless imported into peroxisomes (Kumar et al., 2016; Yang et al., 2001). These proteins use “piggy-backing” with a PTS-containing protein and therefore utilize the capacity of peroxisomes to import oligomers (Kumar et al., 2016).
Compared to matrix proteins, the peroxisomal membrane protein (PMP) family consists of not more than 10 specific proteins including proteins involved in peroxisomal biogenesis or import of matrix proteins and substrates (Antonenkov and Hiltunen, 2012). For mammalians, it is generally accepted that peroxisomes use three proteins – PEX3, PEX19 and PEX16 – for the insertion of PMPs into the peroxisomal membrane (Giannopoulou et al., 2016). However, there is still conflicting information about the mechanism of import of membrane proteins into peroxisomes resulting in two proposed models for PMP import: 1) the ER-derived peroxisome model and 2) the direct post-translational insertion of PMPs (Fig. 7).
According to the first model, PMPs are co-translationally secreted into the ER where they cluster in distinct ER subdomains and finally bud off in preperoxisomal vesicles (ppV) (Tabak et al., 2013). This hypothesis was established more than 30 years ago when several researchers observed a potential involvement of the ER in peroxisomal biogenesis (see Chapter 1.4.3.3) (Lazarow and Fujiki, 1985). Today it is known that PEX19, a cytosolic PMP receptor, recognizes the membrane peroxisomal targeting signal (mPTS, a cluster of basic and hydrophobic residues plus a transmembrane segment (Giannopoulou et al., 2016)) of PMPs and is required for the budding of ER-derived ppVs (Lam et al., 2010). This PMP import model is further supported by the finding that the membrane protein PEX16 lacking its mPTS remains in the ER rather than being transported to peroxisomes (Kim et al., 2006). However, it remains unclear, how PEX19 mediates the budding. One possibility might be the recruitment of vesicle-forming factors (Kim and Hettema, 2015). The ppVs fuse with mature peroxisomes or with each other forming the docking/translocation machinery e.g. for matrix protein import (Agrawal et al., 2016).
In contrast, the alternative model describing import without direct involvement of the ER, involves PEX19 as PMP receptor, recognizing the mPTSs on PMPs and shuttling them from the cytosol to the peroxisomal membrane (Fang et al., 2004). There, PEX19 interacts with the membrane-anchored PEX3 which deforms the peroxisomal membrane and therefore allows membrane insertion (Chen et al., 2014). Markedly, PEX3 is imported into the peroxisomal membrane independently of PEX19; it belongs to the ER-derived PMP family and is first imported in a PEX16-dependent manner into the ER before being targeted to the peroxisomal membrane (Aranovich et al., 2014).
