The GPR37/PAEL Receptor – Finding a Way to the Cell Surface reticulum (ER) and then transported to the cell membrane.14 Unfolded


Pael-R is normally translocated across the ER membrane into the cytosol and degraded through a parkin-dependent ubiquitin– proteasome pathway, which suppresses cell death induced by the accumulation of unfolded Pael-R.9–11


In detail, parkin eliminates unfolded Pael-R in co-operation with the molecular chaperone 70kDa heat shock protein (Hsp70) and a U-box protein known as ct of Hsp70 interacting protein (CHIP).10,11,15


U-box was initially identified in the yeast E4 Ufd2 protein and contains a really interesting new gene (RING) finger fold.16


ubiquitinates unfolded proteins.18


is structurally similar to RING finger motifs and functionally helps in protein degradation.17


Figure 1: Alignment and Structure of Pael-R Ligand-binding domain


A N


The The RING finger fold G-protein- coupling domain


The U-box in CHIP shows E3 activity and Hsp70 plays a role in ensuring


correct folding and intracellular localisation of newly synthesised polypeptides.19


binding to parkin, leading to Pael-R ubiquitination.11,15 B


Moreover, Hsp70 exerts protective properties by When unfolded


Pael-R is translocated to the cytosol, Hsp70 and Hdj2 (ER-associated Hsp40) bind to unfolded Pael-R and initiate upregulation of CHIP.10,11,15 CHIP facilitates the dissociation of Hsp70 from Pael-R and helps in binding of parkin and ubiquitination of the Pael-R in conjunction with E2 ligases such as Ubc4, Ubc6 and Ubc7 on the ER surface.10,11,15


The aggregation of Pael-R due to ineffective ubiquitination by mutated parkin may be involved in ER oxidative stress, decreased cell viability and neurotoxicity in PD.10,11,15


Overexpression of Pael-R, especially in


the absence of parkin, in cell cultures or in animal models leads to unfolded protein-induced cell death.10,11,15,20,21


In addition, when Pael-R is


pan-neuronally overexpressed in drosophila, dopaminergic neurons show selective degeneration.22


These findings suggest that Pael-R It also appears that human Pael-R


orthologues are overexpressed in neurons of the substantia nigra pars compacta in PD patients.23


overexpression causes the selective degeneration of dopamergic neurons. Importantly, Pael-R is localised in the core of Lewy bodies (a hallmark of PD pathology) and Lewy neuritis and parkin in the halo of Lewy bodies and in neuronal cell bodies.24


Moreover, accumulation of


unfolded Pael-R protein in the insoluble fraction of brains of AR-JP patients10


and the presence of this receptor in the core of Lewy bodies in PD patients24


suggest the involvement of aggregated Pael-R in PD pathology and the role of parkin in degradation of Pael-R.9–11


The poor folding of Pael-R also makes difficult its plasmid- mediated overexpresson in cells25


(see Figure 2). The intrinsic


mechanisms, in addition to parkin-mediated degradation, that may regulate the levels of aggregated Pael-R include ER-associated protein degradation (ERAD), UPR, Hsp and molecular chaperone activation and autophagy. Briefly, the ER controls the maturation of membrane and secretory proteins, where newly synthesised secretory proteins enter the ER and bind to ER chaperones, facilitating proper protein folding.26 Folded proteins then enter the secretory pathway, composed of the Golgi apparatus, and proceed to the plasma membrane. Transmembrane proteins, upon internalisation, can be degraded through the ubiquitin–proteasome pathway. When proteins such as Pael-R are not correctly folded, they undergo degradation by ERAD.11,14,26 The accumulation of unfolded proteins leads to ER stress, which transactivates multiple genes including molecular chaperones and


DRUG DISCOVERY


Control of Pael-R Protein Levels Intrinsic Mechanisms that Control Pael-R Toxicity The Pael-R has been shown to be inherently difficult to fold, which enforces the importance of correct degradation events for misfolded Pael-R.11


A: Schematic representation of Pael-R. B: The human Pael-R (015354) protein is aligned with mouse (Q9QY42) and rat (Q9QYC6). The C-terminus and seven transmembrane domains are represented in blocks.


L C A L C I D R F R A ATNVQMY Y EMI ENC S S T TA K L A L C A L C I D R F R A ATNVQMY Y EMI ENC S S T TA K L A L C A L C I D R F R A ATNVQMY Y EMI ENC S S T TA K L A


RGATSWDLPPPRGGD––TGVIEEAAASGLPLGPPTKPPGAWRWKGAQGKEPSGHLGRREPTASQLFRQT RGATSWDLPPPRGGD– – TGVIEEAAASGLPLGPPTKPPGAWRWKGAQGKEPSGHLGRREPTASQLFRQT


RGATSWDLPPPRGGD– – TGVIEEAAASGLPLGPP LAGPSWDLOAAPGRDPAAGRGAEASAAGPPGP


TKPPGAWRWKGAQGKEPSGHLGRREPTASQLFRQT TRPPGPWRWKGARGQEPS E T LGRGNPTALQL F LQI


LAGPSWDLOAAPGRDPAAGRGAEASAAGPPGPTRPPGPWRWKGARGQEPSETLGRGNPTALQLFLQI RGATSWDLPPPRGGD––TGVIEEAAASGLPLGPPTKPPGAWRWKGAQGKEPSGHLGRREPTASQLFRQT


I S E E E E KGP RGAGI SGR SQEQS V K T V PGASDL F YWP R RAGK LQGSHHK P L S K TANGLAGHEGWY IAL P T S E RGEMS S K RDE I PQGSQEHS V K T E P – – RDL F YWP R K TGQLQGSHY R P S – – – – – – –AVHEGR T LAP P T S E RGEMS S K RDE I PQGSQEHS V K T E P E P RDL F YWP R K TGQLQGSHY R P S – – – – – – –AVHEGR T LAP P


TM1


GRALAQNGSLGEGIHEPGGPRRGNSTNRRVRLKNFYPLTQESYGA SVMCLSVVIFGTIIGNLAVM G R A L A Q N G S L G E G I H E P G G P R R G N T T – R R V R L K N F Y P LT Q E S Y G AY S VMC L S V V I F G T I I G N L AVM


G R A L A Q N G S L G E G I H E P G G P R R G N S T N R R V R L K N F Y P LT Q E S Y G AY S VMC L S V V I F G T I I G N L AVM GRALAQNGSLGEGIHEPGGPRRGNTT–RRVRLKNFYPLTQESYGA SVMCLSVVIFGTIIGNLAVM


GRALAQNGSLGEGIHEPGGPRRGNSTNRRVRLKNFYPLTQESYGA SVMCLSVVIFGTIIGNLAVM G R A L A Q N G S L G E G I H E P G G P R R G N S T N R R V R L K N F Y P LT Q E S Y G AY S VMC L S V V I F G T I I G N L AVM TM2 TM3 TM3


CIVVHNYYMRSISMSLLANLAFWDFLIIFFCLPLVIFHELTKKWLLEDFSCKIVPYIEVASLGVTTFT CIVVHNYYMRSISMSLLANLAFWDFLIIFFCLPLVIFHELTKKWLLEDFSCKIVPYIEVASLGVTTFT CIVVHNYYMRSISMSLLANLAFWDFLIIFFCLPLVIFHELTKKWLLEDFSCKIVPYIEVASLGVTTFT


C I V V H N Y YMR S I SMS L L A N L A FWD F L I I F F C L P LV I F H E LT K KWL L E D F S C K I V P Y I E VA S L G V T T F C I V V H N Y YMR S I SMS L L A N L A FWD F L I I F F C L P LV I F H E LT K KWL L E D F S C K I V P Y I E VA S L G V T T F C I V V H N Y YMR S I SMS L L A N L A FWD F L I I F F C L P LV I F H E LT K KWL L E D F S C K I V P Y I E VA S L G V T T F


TM4


LCALCIDRFRAATNVQMYYEMIENCSSTTAKLAVIWVGALLLALPEVVLRQLSKEDLGFSGRAPAERC LCALCIDRFRAATNVQMYYEMIENCSSTTAKLAVIWVGALLLALPEVVLRQLSKEDLGFSGRAPAERC LCALCIDRFRAATNVQMYYEMIENCSSTTAKLAVIWVGALLLALPEVVLRQLSKEDLGFSGRAPAERC


V IWV G A L L L A L P E V V L RQL S K E D L G F S G R A PA E R C V IWV G A L L L A L P E V V L RQL S K E D L G F S G R A PA E R C V IWV G A L L L A L P E V V L RQL S K E D L G F S G R A PA E R C


TM5


IIKISPDLPDTIYVLALTYDSARLWWYFGCYFCLPTLFTITCSLVTSRKIRKAEKACTRGNKRQIQLE IIKISPDLPDTIYVLALTYDSARLWWYFGCYFCLPTLFTITCSLVTSRKIRKAEKACTRGNKRQIQLE IIKISPDLPDTIYVLALTYDSARLWWYFGCYFCLPTLFTITCSLVTSRKIRKAEKACTRGNKRQIQLE


I I K I S P D L P D T I Y V L A LT Y D S A R LWWY F G C Y F C I I K I S P D L P D T I Y V L A LT Y D S A R LWWY F G C Y F C I I K I S P D L P D T I Y V L A LT Y D S A R LWWY F G C Y F C


TM6 SQMNCTVVALTILYGFCIIPENICNIVT


L P T L F T I T C S LV T S K I R K A E K A C T R G N K R Q I Q L E L P T L F T I T C S LV T S K I R K A E K A C T R G N K R Q I Q L E L P T L F T I T C S LV T S K I R K A E K A C T R G N K R Q I Q L E


R R R


TM7


S QMN C T V VA LT I LY G F C I I P E N I C N I V TAYMATGVSQQTMDLLNIISQFLLFFKSCVTPVLLFCLCKPMAT G V S Q Q TMD L L N I I S Q F L L F F K S C V T P V L L F C L C K P SQMNCTVVALTILYGFCIIPENICNIVT


S QMN C T V VA LT I LY G F C I I P E N I C N I V TAYMATGVSQQTMDLLNIISQFLLFFKSCVTPVLLFCLCKPMAT G V S Q Q TMD L L N I I S Q F L L F F K S C V T P V L L F C L C K P SQMNCTVVALTILYGFCIIPENICNIVT


S QMN C T V VA LT I LY G F C I I P E N I C N I V TAYMATGVSQQTMDLLNIISQFLLFFKSCVTPVLLFCLCKPMAT G V S Q Q TMD L L N I I S Q F L L F F K S C V T P V L L F C L C K P ct–terminal PDZ–motif


F S RAFME C C C C C C E E C IQK S S T V T SDDNDNE Y T T E L E L S P F S T I R R EMS T FAS VGTHC Human F S RAFME C C C C C C E E C IQK S S T V T SDDNDNE Y T T E L E L S P F S T I R R EMS T FAS VGTHC Mouse F S RAFME C C C C C C E E C IQK S S T V T SDDNDNE Y T T E L E L S P F S T I R R EMS T FAS VGTHC Rat


FSRAFMECCCCCCEECIQKSSTVTSDDNDNEYTTELELSPFSTIRREMSTFASVGTHC FSRAFMECCCCCCEECIQKSSTVTSDDNDNEYTTELELSPFSTIRREMSTFASVGTHC FSRAFMECCCCCCEECIQKSSTVTSDDNDNEYTTELELSPFSTIRREMSTFASVGTHC


015354 Q9QY42 Q9QYC6


T T T


ISEEEEKGPRGAGISGRSQEQSVKTVPGASDLFYWPRRAGKLQGSHHKPLSKTANGLAGHEGWYIALP TSERGEMSSKRDEIPQGSQEHSVKTEP––RDLFYWPRKTGQLQGSHYRPS–––––––AVHEGRTLAPP TSERGEMSSKRDEIPQGSQEHSVKTEPEPRDLFYWPRKTGQLQGSHYRPS–––––––AVHEGRTLAPP


ERAD-associated molecules, leading to UPR and, possibly, apoptosis.27 The overexpression of Pael-R may also induce the cellular autophagic pathway.28


Autophagy induced by Pael-R overexpression plays an important role in clearing protein aggregates and prevents the degeneration of Pael-R-expressing neurons.28


Interestingly, the levels of


both ERAD and autophagic markers were found to be altered in brain extracts of Pael-R knockout mice.28


Potential Targets to Control Pael-R Toxicity To date, in addition to parkin there are four further proteins that have been shown to regulate Pael-R toxicity and could be potential drug targets; these are summarised below.


•


Human homologue of yeast Hrd1p (HRD1) is an E3 ligase involved in ERAD and expressed in dopaminergic neurons of the substantia nigra. HRD1 directly co-localises with Pael-R in the ER and promotes the ubiquitination and degradation of Pael-R in the ER, thus suppressing Pael-R-induced cell death.29


37


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