WO2017046592A1 - Cibles en aval de pink1 - Google Patents

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Publication number
WO2017046592A1
WO2017046592A1 PCT/GB2016/052884 GB2016052884W WO2017046592A1 WO 2017046592 A1 WO2017046592 A1 WO 2017046592A1 GB 2016052884 W GB2016052884 W GB 2016052884W WO 2017046592 A1 WO2017046592 A1 WO 2017046592A1
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pink1
rab8a
phosphorylation
ser
disease
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PCT/GB2016/052884
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English (en)
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Miratul Muqit
Matthias Trost
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University Of Dundee
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G01N33/6896—Neurological disorders, e.g. Alzheimer's disease
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/28—Neurological disorders
    • G01N2800/2835—Movement disorders, e.g. Parkinson, Huntington, Tourette
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

  • the present invention relates to a biomarker for Parkinson's disease.
  • the biomarker and products associated with the biomarker may be used to assist diagnosis or to assess onset and/or development or progression of Parkinson's disease.
  • the invention also relates to use of the biomarker in evaluation of drug treatments, drug screening or drug development in the field of Parkinson's disease and Parkinson's disease related disorders.
  • Parkinson's disease is a degenerative disease of the central nervous system which mainly affects the motor system.
  • the motor symptoms of this disease (which include tremor, bradykinesia and rigidity) result from the death of cells present in the substantia nigra which generate the neurotransmitter dopamine.
  • Treatments of PD include medications such as dopamine agonists, levodopa and monoamine oxidase-B inhibitors.
  • these treatments can be useful at improving the early motor systems, such treatments do not affect underlying disease progression and eventually become ineffective.
  • PINK1 and Parkin null flies exhibit significant mitochondrial defects and that PINK1 lies genetically upstream of Parkin (Clark et al, 2006; Park et al, 2006).
  • PINK1 is activated in response to mitochondrial depolarization and this stimulates recruitment of Parkin, a cytosolic protein, to depolarized mitochondria where it ubiquitylates multiple mitochondrial substrates to trigger removal of mitochondria by autophagy (also known as mitophagy) (Geisler et al, 2010; Matsuda et al, 2010; Narendra et al, 2008; Narendra et al, 2010; Vives-Bauza et al, 2010).
  • PINK1 directly phosphorylates Parkin at Serine65 (Ser 65 ) within its ubiquitin-like (Ubl) domain (Kondapalli et al, 2012; Shiba-Fukushima et al, 2012) and ubiquitin at an equivalent Ser 65 residue (Kane et al, 2014; Kazlauskaite et al, 2014; Koyano et al, 2014).
  • PINK1 is normally imported to mitochondria where its levels are kept low due to constitutive cleavage by mitochondrial proteases (Deas et al, 201 1 ; Jin et al, 2010; Meissner et al, 201 1 ) and proteasomal degradation via the N-end rule pathway (Yamano & Youle, 2013).
  • PINK1 import via the TOM40 and TIM23 complexes is blocked and PINK1 is able to escape proteolytic cleavage and accumulate at the outer mitochondrial membrane (OMM) (Narendra et al, 2010) where it becomes catalytically active as judged by PINK1 autophosphorylation and phosphorylation of substrates (Kondapalli et al, 2012; Okatsu et al, 2012).
  • OMM outer mitochondrial membrane
  • Phosphorylation of amino acid residues of PINK1 , particularly Thr 257 has therefore been described as a biomarker for PD (as described in PCT/GB2013/050926).
  • PINK1 and Parkin in a common pathway fits seamlessly with clinical observations that PD patients with PINK1 and Parkin mutations have similar phenotypes (Khan et al, 2002).
  • analysis of rat knockout models of PINK1 and Parkin indicate the existence of additional PINK1 -dependent phosphorylation sites: PINK1 knockout rats exhibited progressive neurodegeneration whereas Parkin knockout rats remained unaffected suggesting that PINK1 may regulate additional proteins that are essential for neuronal integrity and survival in the mammalian brain (Dave et al, 2014).
  • PINK1 downstream signalling may in part be distinct from Parkin (Zhang et al, 2013).
  • the present invention provides biological markers ("biomarkers”) relating to PD.
  • biomarkers biological markers relating to PD.
  • the present invention is based, in part, on studies by the inventors into phosphorylation targets of PINK1 .
  • the studies of the inventors revealed that PINK1 regulates the phosphorylation of a highly conserved residue, Serine 1 1 1 (Ser 111 ) of a family of Rab GTPases, namely Rab8A, Rab8B and Rab13 and equivalent conserved residues of other Rab GTPase family members, for example Ser 114 of Rab1 A.
  • an in vitro method of diagnosing Parkinson's disease, monitoring of progression of Parkinson's disease, assessing response to therapy and/or screening drugs for treating Parkinson's disease comprising detecting phosphorylation of amino acid residues of Rab GTPases.
  • the biomarkers of the present invention include phosphorylation of amino acid residues of the PINK1 targets Rab8A, Rab8B, Rab13 and/or RablA.
  • the present invention includes identification of phosphorylation of the Ser 111 residue of Rab8A, Rab8B and Rab13 (numbering according to GenBank: CR542274, BC020654 and BC000799) or equivalent conserved residues of other Rab GTPase family members, for example Ser 114 of RablA or Ser 111 of RabI B (numbering according to GenBank: CR536488.1 and CR533462.1 ) (see Figure 17 for example).
  • the invention will further comprise comparing the detected phosphorylation level with the phosphorylation level in an appropriate control.
  • the appropriate control will be dependent upon the method. For example, when diagnosing PD, the control might be a standard level or reference range in a non-PD subject. Alternatively, when monitoring progression of PD, the control might be the phosphorylation level in a biological sample obtained from the subject at an earlier time. It will be well within the capabilities of the skilled person to determine appropriate controls.
  • comparison with the phosphorylation level in a control may not be required.
  • the level of phosphorylation in a control for example a non-PD subject
  • a phosphorylation detector may not indicate a positive result below a certain threshold. In these instances, if phosphorylation is detected in a test subject, a change in the level of phosphorylation can be confirmed without a comparison step.
  • the invention provides a method for determining whether a subject is more likely than not to have PD, or is more likely to have PD than to have another disease.
  • the method of the present invention may be performed by analysing a biological sample, such as serum or CSF from the subject; measuring the level of phosphorylation of at least one of the biomarkers in the biological sample; and optionally comparing the measured phosphorylation level with a standard level or reference range.
  • a biological sample such as serum or CSF from the subject
  • measuring the level of phosphorylation of at least one of the biomarkers in the biological sample and optionally comparing the measured phosphorylation level with a standard level or reference range.
  • the standard level or reference range is obtained by measuring the same marker or markers in a normal control or, more preferably, a set of normal controls. Depending upon the difference between the measured level and the standard level or reference range, the patient can be diagnosed as having or being predisposed to developing PD, or as not having PD.
  • a standard level or reference range is specific to the biological sample at issue.
  • a standard level or reference range for the marker in serum that is indicative of PD would be expected to be different from the standard level or reference range (if one exists) for that same marker in CSF, urine or another tissue, fluid or compartment.
  • references herein to measuring biomarkers will be understood to refer to measuring the level of phosphorylation of the biomarker.
  • references herein to comparisons between a marker phosphorylation measurement level and a standard level or reference range will be understood to refer to such levels or ranges for the same type of biological sample.
  • a method for monitoring a PD patient over time may be performed by analysing a biological sample, such as serum or CSF, from the subject at a certain time; measuring the phosphorylation level of at least one of the biomarkers in the biological sample; and comparing the measured phosphorylation level with the phosphorylation level measured with respect to a biological sample obtained from the subject at an earlier time.
  • the invention provides a method for conducting a clinical trial to determine whether a candidate drug is effective in treating PD.
  • the method may be performed by analysing a biological sample from each subject in a population of subjects diagnosed with PD, and measuring the phosphorylation level of at least one of the biomarkers in the biological samples. Then, a dose of a candidate drug is administered to one portion or sub-population of the same subject population ("experimental group") while a placebo is administered to the other members of the subject population (“control group”). After drug or placebo administration, a biological sample is acquired from the experimental and control groups and the same assays are performed on the biological samples as were previously performed to obtain phosphorylation measurement values.
  • the candidate drug is effective.
  • the relative efficacy of two different drugs or other therapies for treating PD can be evaluated using this method by administering the drug or other therapy in place of the placebo.
  • the methods of the present invention may be used to evaluate an existing drug, being used to treat another indication, for its efficacy in treating PD (e.g. by comparing the efficacy of the drug relative to one currently used for treating PD in a clinical trial, as described above).
  • the present invention also provides molecules that specifically bind to the phosphorylated or unphosphorylated residue, or region comprising the residue, such as an antibody or antibody fragment, or aptamer.
  • marker specific reagents have utility in isolating the markers and in detecting the presence of the markers, e.g. in immunoassays.
  • the molecule should bind to the epitope of interest (i.e. a region of a PINK1 target which comprises the phosphorylated or unphosphorylated residue) with binding affinity sufficient to prevent dissociation of the molecule from the epitope when standard washing steps are performed, for example in immunoassays.
  • the molecules may bind with sufficient specificity that the molecules can be used to isolate molecules bearing the biomarkers of interest from a population of molecules.
  • the skilled person with their common general knowledge will be able to identify molecules with sufficient binding affinity and specificity.
  • region comprising the residue will be understood by the skilled person to infer that the molecule does not have to bind only to the epitope of interest (i.e. the phosphorylated or unphosphorylated residue) but may also form interactions with amino acid residues surrounding the phosphorylated or unphosphorylated residue.
  • the biomarkers may be used for diagnostic purposes. However, they may also be used for therapeutic, drug screening and patient stratification purposes (e.g. to group patients into a number of "subsets" for evaluation).
  • the present invention includes all methods relying on correlations between the biomarkers described herein and the presence of PD.
  • the invention provides methods for determining whether a candidate drug is effective at treating PD by evaluating the effect it has on the biomarker values.
  • the term "effective" is to be understood broadly to include reducing or alleviating the signs or symptoms of PD, improving the clinical course of the disease, decreasing the number or severity of exacerbations, or reducing any other objective or subjective indicia of the disease.
  • Different drugs, doses and delivery routes can be evaluated by performing the method using different drug administration conditions. The method may also be used to compare the efficacy of two different drugs or other treatments or therapies for PD.
  • Phosphorylation levels may be measured using any suitable stain or dye that recognises phosphor groups associated with proteins or peptides, for example Pro-Q Diamond or phosphor specific antibodies.
  • the phosphorylation levels can also be measured after purification with different affinity columns such as IMAC or any other phosphor-binding surfaces. It is expected that the levels of phosphorylation of the biomarkers described herein may be measured in combination with other signs, symptoms and clinical tests of PD, and/or other PD biomarkers reported in the literature (for example Parkin Ser 65 phosphorylation, as described in PCT/GB2013/050926). Likewise, more than one of the biomarkers of the present invention may be measured in combination. Measurement of the phosphorylation of the biomarkers of the invention along with any other markers known in the art (including those not specifically listed herein) falls within the scope of the present invention.
  • the method of the present invention may be used to determine whether a subject is more likely than not to have PD, or is more likely to have PD than to have another disease, based on the difference between the measured and standard level or reference range of the biomarker.
  • a patient with a putative diagnosis of PD may be diagnosed as being "more likely” or “less likely” to have PD in light of the information provided by a method of the present invention.
  • the method of the present invention may therefore be used to assist a clinician with diagnosis of PD.
  • the biological sample may be of any tissue or fluid.
  • the sample is a CSF or serum sample, but other biological fluids or tissue may be used.
  • Possible biological fluids include, but are not limited to, plasma, urine and neural tissue.
  • a CSF biomarker in itself may be particularly useful for early diagnosis of disease.
  • molecules initially identified in CSF may also be present, presumably at lower concentrations, in more easily obtainable fluids such as serum and urine.
  • Such biomarkers may be valuable for monitoring all stages of the disease and response to therapy.
  • Serum and urine also represent preferred biological samples as they are expected to be reflective of the systemic manifestations of the disease.
  • the level of a marker may be compared to the level of another marker or some other component in a different tissue, fluid or biological "compartment.”
  • a differential comparison may be made of a marker in CSF and serum. It is also within the scope of the invention to compare the level of a marker with the level of another marker or some other component within the same compartment.
  • Phosphorylation measurements can be of (i) a biomarker of the present invention, (ii) a biomarker of the present invention and another factor known to be associated with PD (e.g. a PET scan); (iii) a plurality of biomarkers comprising at least one biomarker of the present invention and at least one biomarker reported in the literature; or (iv) any combination of the above.
  • the amount of change in a biomarker level may be an indication of the relative likelihood of the presence of the disease.
  • the present invention provides phosphorylated biomarkers that the present inventors have shown to be indicative of PD in a subject. It is to be understood that any correlations between biological sample measurements of these biomarkers and PD, as used for diagnosis of the disease or evaluating drug effect, are within the scope of the present invention.
  • phosphorylated biomarker levels are measured using conventional techniques which will be well known by the skilled person.
  • a wide variety of techniques are available, for example, mass spectrometry, chromatographic separations, 2-D gel separations, binding assays (e.g. immunoassays), competitive inhibition assays, and so on.
  • Any effective method in the art for measuring the level of a protein or low molecular weight marker is included in the invention.
  • the skilled person would be capable of determining which method would be most appropriate for measuring a specific marker.
  • a robust ELISA assay may be best suited for use in a physician's office while a measurement requiring more sophisticated instrumentation may be best suited for use in a clinical laboratory. Regardless of the method selected, it is important that the measurements be reproducible.
  • the phosphorylated markers of the present invention can be measured by mass spectrometry, which allows direct measurements of analytes with high sensitivity and reproducibility.
  • mass spectrometric methods are available and could be used to accomplish the measurement.
  • Electrospray ionization (ESI) allows quantification of differences in relative concentration of various species in one sample against another; absolute quantification is possible by normalization techniques (e.g., using an internal standard).
  • Matrix-assisted laser desorption ionization (MALDI) or the related SELDI® technology (Ciphergen, Inc.) also could be used to make a determination of whether a marker was present, and the relative or absolute level of the marker.
  • mass spectrometers that allow time-of-flight (TOF) measurements have high accuracy and resolution and are able to measure low abundant species, even in complex matrices like serum or CSF.
  • quantification can be based on derivatization in combination with isotopic labelling, referred to as isotope coded affinity tags ("ICAT").
  • ICAT isotope coded affinity tags
  • a specific amino acid in two samples is differentially and isotopically labelled and subsequently separated from peptide background by solid phase capture, wash and release. The intensities of the molecules from the two sources with different isotopic labels can then be accurately quantified with respect to one another.
  • one- and two-dimensional gels have been used to separate proteins and quantify gels spots by silver staining, fluorescence or radioactive labelling. These differently stained spots have been detected using mass spectrometry, and identified by tandem mass spectrometry techniques.
  • the phosphorylated markers are measured using mass spectrometry in connection with a separation technology, such as liquid chromatography-mass spectrometry or gas chromatography-mass spectrometry. It is preferable to couple reverse-phase liquid chromatography to high resolution, high mass accuracy ESI time-of-flight (TOF) mass spectroscopy. This allows spectral intensity measurement of a large number of biomolecules from a relatively small amount of any complex biological material without sacrificing sensitivity or throughput. Analysing a sample will allow the marker (specified by a specific retention time and m/z) to be determined and quantified. As will be appreciated by the skilled person, many other separation technologies may be used in connection with mass spectrometry.
  • a separation technology such as liquid chromatography-mass spectrometry or gas chromatography-mass spectrometry. It is preferable to couple reverse-phase liquid chromatography to high resolution, high mass accuracy ESI time-of-flight (TOF) mass spectroscopy. This allows
  • separations may be performed using custom chromatographic surfaces (e.g. a bead on which a marker specific reagent has been immobilised). Molecules retained on the media subsequently may be eluted for analysis by mass spectrometry.
  • Analysis by liquid chromatography-mass spectrometry produces a mass intensity spectrum, the peaks of which represent various components of the sample, each component having a characteristic mass- to-charge ratio (m/z) and retention time (r.t).
  • the presence of a peak with the m/z and retention time of a biomarker indicates that the marker is present.
  • the peak representing a marker may be compared to a corresponding peak from another spectrum (e.g. from a control sample) to obtain a relative measurement.
  • Any normalisation technique in the art e.g. an internal standard
  • deconvoluting software is available to separate overlapping peaks.
  • the retention time depends to some degree on the conditions employed in performing the liquid chromatography separation.
  • the level of phosphorylation of the markers may be determined using a standard immunoassay, such as sandwiched ELISA using matched antibody pairs and chemiluminescent detection.
  • a standard immunoassay such as sandwiched ELISA using matched antibody pairs and chemiluminescent detection.
  • Commercially available or custom monoclonal or polyclonal antibodies are typically used.
  • the assay can be adapted for use with other reagents that specifically bind to the marker such as Affibody polypeptides. Standard protocols and data analysis are used to determine the marker concentrations from the assay data.
  • markers of the invention include selective reaction monitoring/targeted quantitative proteomics and antibody mediated immunofluorescence, immunohistochemistry or immunoblotting of cerebrospinal fluid, brain biopsy material and/or skin fibroblasts converted to neurons using iPS technology.
  • a number of the assays discussed above employ a reagent that specifically binds to the phosphorylated marker ("marker specific reagent"). Any molecule that is capable of specifically binding to a marker is included within the invention.
  • the marker specific reagents are antibodies or antibody fragments.
  • the marker specific reagents are non-antibody species.
  • a marker specific reagent may be an enzyme for which the marker is a substrate.
  • the marker specific reagents may recognize any epitope of the targeted markers.
  • phosphorylation of only one biomarker is measured, then that value must increase to indicate drug efficacy. If more than one biomarker is measured, then drug efficacy can be indicated by change in only one biomarker, all biomarkers, or any number in between. In some embodiments, multiple markers are measured, and drug efficacy is indicated by changes in multiple markers. Phosphorylation measurements can be of both biomarkers of the present invention and other measurements and factors associated with PD (e.g. measurement of biomarkers reported in the literature and/or other diagnostic techniques). Furthermore, the amount of change in a biomarker phosphorylation level may be an indication of the relative efficacy of the drug.
  • biomarkers of the invention can also be used to examine dose effects of a candidate drug.
  • dose effects of a candidate drug There are a number of different ways that varying doses can be examined. For example, different doses of a drug can be administered to different subject populations, and phosphorylation measurements corresponding to each dose analysed to determine if the differences in the inventive biomarkers before and after drug administration are significant. In this way, a minimal dose required to effect a change can be estimated.
  • results from different doses can be compared with each other to determine how each biomarker behaves as a function of dose.
  • administration routes of a particular drug can be examined.
  • the drug can be administered differently to different subject populations, and phosphorylation measurements corresponding to each administration route analysed to determine if the differences in the inventive biomarkers before and after drug administration are significant. Results from the different routes can also be compared with each other directly.
  • kits for diagnosing PD, monitoring progression of the disease and assessing response to therapy comprising at least one marker specific reagent.
  • a kit may comprise an antibody which is capable of specifically binding the phosphorylated or unphosphorylated residues of a PINK1 target as identified herein.
  • the kit may comprise two antibodies, one which is capable of binding a phosphorylated residue as described herein and another which is capable of binding an unphosphorylated residue as described herein.
  • Each antibody may be differentially labelled in order to permit a user to discern which antibody has bound the target and hence whether or not the target is phosphorylated or not.
  • kits may further comprise a container for a sample collected from a subject.
  • a container for a sample collected from a subject In developing such kits, it is within the competence of the skilled person to perform validation studies that would use an optimal analytical platform for each marker. For a given marker, this may be an immunoassay or mass spectrometry assay. Kit development may require specific antibody development, evaluation of the influence (if any) of matrix constituent ("matrix effects"), and assay performance specifications. It may turn out that a combination of two or more markers provides the best specificity and sensitivity, and hence utility for monitoring the disease.
  • FIG. 1 SILAC phosphoproteomic approach for identification of PINK1- dependent targets.
  • Flp-ln TRex HEK293 cells stably expressing FLAG-alone were cultured in unlabeled (R0K0) medium, kinase inactive (Kl; D384A) PINK1 -FLAG were 'medium' (R6K4) labeled, and wild-type (WT) PINK1 -FLAG cells were 'heavy' (R10K8) labeled and using SILAC media containing the respective isotopes. All conditions were treated with 10 ⁇ CCCP for 3 hours and subjected to membrane fractionation.
  • FIG. 2 Analysis of PINK1 -regulated phosphoproteome and identification of Ser phospho-peptides of Rab GTPases.
  • A Volcano plot highlighting significantly (p,0.05, >3-fold change) up-regulated and down-regulated phosphopeptides identified in each screen.
  • Rab GTPases are labelled.
  • B Sequence alignment of Ser 111 phosphorylation site in Rab8A, Rab8B and Rab13 orthologs from mammals to drosophila shows high conservation around the Ser 111 phosphorylation site (asterisk).
  • Figure 3 Analysis of PINK1 dependent phospho-proteins.
  • A Pie chart analysis showing subcellular localization of PINK1 up-regulated phospho-proteins. Membrane- bound proteins make up more than half of the regulated phosphoproteins.
  • B PINK1 up-regulated phospho-proteins sub-grouped according to cell localization. Magenta hexagon: Serine phosphorylation residue; Blue hexagon: Threonine phosphorylation residue; Yellow hexagon: Tyrosine phosphorylation residue.
  • C Table of PINK1 - dependent phosphopeptides that were up-regulated across all four replicates
  • Figure 4 Analysis of PINK1-regulated phosphoproteome. Comparison of average ratio of phosphopeptide between WT PINK1/KI PINK1 and WT PINK/empty vector.
  • Figure 5 Rab8A, Rab8B and Rab13 Ser phosphorylation are regulated by PINK1 upon CCCP treatment.
  • A-C Confirmation by mass spectrometry that Rab8A (A), Rab8B (B) and Rab13 (C) Ser 111 is phosphorylated upon PINK1 activation after CCCP treatment.
  • PINK1 -FLAG Fip-!n TRex HEK293 cells expressing empty-FLAG, WT PINK1 -FLAG, and Ki (D384A) PINK1 -FLAG were transfected either with HA- RabSA (A), HA-Rab8B (B) or HA-Rab13 (C) induced with doxycycline and stimulated with 10 ⁇ of CCCP for 3 hours.
  • Whole cell lysates (10 mg) were immunoprecipitated with anti-HA agarose, resolved by SDS-PAGE and stained with Colloidal Coomassie blue (second panel).
  • Coomassie-stained bands migrating with expected molecular mass of HA-Rabs were excised, in-gel digested with trypsin and subjected to high performance liquid chromatography with LC-MS/MS on an LTQ-Orbitrap mass spectrometer.
  • Upper panel shows the extracted ion chromatogram (XiC) analysis of Ser 111 containing phosphopeptide (SIKENApSAGVER) with the combined signal intensity of the 2 + and 3 + forms of the peptide indicated on the y-axis. Note that the Ser 11 phosphopeptide was only detected in samples from WT PINK1 -FLAG expressing cells following CCCP stimulation.
  • D Characterization of RabSA, RabSB and Rab13 phospho-Ser 111 antibodies.
  • Flp-ln TRex HEK293 ceils expressing empty-FLAG, WT PINK1 -FLAG, and Kl (D384A) PINK1 -FLAG were transfected with either WT or Ser11 1 Ala mutant (S1 11 A) HA-RabSA, HA-Rab8B or HA-Rab13 induced with doxycycline and stimulated with 10 ⁇ of CCCP for 3 hours.
  • Whole cell lysates (0.25 mg) were immunoprecipitated with anti-HA agarose and immunoblotted with RabSA, RabSB or Rab13 phospho-Ser 111 antibodies. Part of the immunoprecipitates were used to immunoblot for HA-antibody as loading controls.
  • FIG. 6 Endogenous PINK1 regulates Rab8A, Rab8B and Rab13 Ser 111 phosphorylation.
  • WT and PINK1 KO HeLa cells were transfected with either WT or Ser1 1 1Ala (S1 1 1A) mutant constructs of HA-Rab8A, HA-Rab8B or HA-Rab13.
  • Some PINK1 KO HeLa cells were reintroduced with PINK1 by transfection of WT PINK1 - 3xFLAG or Kl (D384A) PINK1 -3xFLAG as indicated.
  • WT and PINK1 KO HeLa cells were treated with DMSO as a vehicle control or CCCP for 20 hours.
  • Other PINK1 KO HeLa cells were reintroduced with PINK1 by transfection of WT PINK1 -3xFLAG or Kl (D384A) PINK1 - 3xFLAG as indicated for at least 24 hours before CCCP treatment.
  • Whole cell lysates (1 mg) were immunoprecipitated with anti-Rab8A (from Cell Signaling Technology) pre- bound with protein A agarose followed by immunoblot with Rab8A phospho-Ser 111 antibody. Part of the immunoprecipitates were used to immunoblot with anti-total Rab8A antibody (from Sigma) as loading controls.
  • FIG. 7 Endogenous PINK1 regulates endogenous Rab8A Ser phosphorylation in HEK293 cells.
  • HEK293 cells were treated with DMSO vehicle control or CCCP for 24 hours.
  • Whole cell lysates (1 mg) were immunoprecipitated with anti-Rab8A (from Cell Signaling Technology) pre-bound with protein A agarose followed by immunoblot with Rab8A phospho-Ser 111 antibody.
  • Part of immunoprecipitates were used to immunoblot with anti-Rab8A antibody (from Sigma) as loading controls.
  • FIG. 8 Rab8A Ser 111 phosphorylation is abolished in PD patient PINK1 fibroblasts and PINK1 knockout mouse embryonic fibroblasts (MEFs)
  • Primary skin fibroblasts were derived from a patient with homozygous PINK1 Q456X mutations or unaffected control. Cells were incubated with DMSO or CCCP for 20 hours and whole cell lysates (1 mg) were immunoprecipitated with anti-Rab8A antibody conjugated to protein A agarose and immunoblotted with total or Rab8A phosphor- Ser 111 antibody.
  • Lysates (1 mg) were also subjected to immunoprecipitation with polyclonal anti-PINK1 antibody and immunoblotted with monoclonal PINK1 antibody. Equal loading of protein extracts was confirmed by GAPDH.
  • B Absence of Rab8A Ser 111 phosphorylation in PINK1 knockout MEFs. MEFs were derived from PINK1 knockout embryos or wildtype controls. Cells were incubated with DMSO or CCCP for 20 hours and whole cell lysates (1 mg) were immunoprecipitated with anti-Rab8A antibody conjugated to protein A agarose and immunoblotted with total or Rab8A phosphor-Ser 111 antibody.
  • Mitochondrial enriched extracts (mitochondrial lysate) were incubated with ubiquitin-binding resins derived from His-Halo-Ubiquilin1 UBA domain tetramer (UBA UBWLN1 ). Captured ubiquitylated proteins were subject to immunoblotting with CISD1 and Mitofusin2 antibodies. Mitochondrial lysate and total lysate were also subjected to immunoblotting with indicated antibodies for loading and protein expression controls. Phospho-Ser 111 Rab8A was detected after Rab8A immunoprecipitation from 200 ⁇ g of mitochondrial lysate with anti-Rab8A antibody (from Cell Signaling Technology).
  • Figure 10 Evidence that Rab8A Ser is not a direct substrate of PINK1.
  • Ubiquitin, Parkin, Rab8A and RabIA phosphosites adopt two distinct conformations.
  • Ser 65 in Ubiquitin (blue) and Parkin (magenta) follows a ⁇ -turn, before the start of the 5th ⁇ -strand of the EF-hand domain.
  • Ser 114 in Rab 1A (green) and Ser 111 in Rab 8A (ochre) lie after a C-terminal helix cap just before the start of a ⁇ -turn.
  • Representative three-dimensional structures are superimposed by C-a positions for the observed phosphosites and their sequence neighbours. Side chains for observed sites are shown as sticks, and ribbons depict backbone and secondary structure.
  • Flp-ln TRex HEK293 cells expressing WT PINK1 -FLAG were transfected with either WT or Ser1 1 1Ala (S1 1 1A) mutant HA-Rab8A, HA-Rab8B or HA-Rab13, induced with doxycycline and stimulated with CCCP for the indicated time.
  • Flp-ln TRex HEK293 cells expressing WT PINK1 -FLAG were transfected with either WT or Ser 65 alanine (S65A) mutant Parkin.
  • Whole cell lysates (0.25 mg) were immunoprecipitated with anti-HA agarose and immunoblotted with indicated phospho-Ser 111 antibodies.
  • FIG. 12 Rab8A Ser 111 phosphorylation impairs Rabin8 catalysed GDP exchange in vitro.
  • A Crystal structure of Rab8A in complex with guanine exchange factor (GEF) Rabin8 (Guo et al, 2013).
  • GEF guanine exchange factor
  • Left panel Rabin8 is shown in surface representation and Rab8A in cartoon representation.
  • Right panel Rabin8 is shown in cartoon representation and Rab8A is shown in surface representation.
  • the hydroxyl group of residue Ser 111 is shown as a yellow sphere.
  • B Rabin8-catalyzed mant-GDP release from mant-GDP loaded WT, S1 1 1A and S1 1 1 E mutants of Rab8A.
  • Rab8A KO HeLa cells were transfected with wildtype (WT), S1 1 1 E or S1 1 1A HA-Rab8A.
  • Whole cell lysates (1 mg) were immunoprecipitated with anti-HA agarose and immunoblotted with Rabin8 or anti-HA antibody. Lysates were immunoblotted with Rabin8 or anti-HA antibody to confirm equivalent expression of Rabin8 and WT and mutant HA-Rab8A in extracts.
  • B Decreased S1 1 1 E Rab8A association with Rabin8 in cells.
  • Rab8A KO HeLa cells were co-transfected with GFP-Rabin8 and wildtype (WT), S11 1 E or S1 1 1 A HA-Rab8A.
  • Whole cell lysates (1 mg) were immunoprecipitated with GFP binder sepharose resin and immunoblotted with anti-HA and anti-GFP antibodies. Lysates were immunoblotted with anti-GFP and anti-HA antibody to confirm equivalent expression of GFP-Rabin8 and HA-Rab8A across all extracts.
  • Figure 15 Comparative analysis of human Rab8 and Rabin8 and yeast orthologues Ypt1/Sec4 and Sec2.
  • A Multiple sequence alignment of yeast Ypt1 , Sec4 and human Rab8A to assess conservation of Rab8A Ser1 1 1 (Ser 111 ) phosphosite. Alignment is shaded according to BLOSUM62 similarity, and Ser 111 site is marked with an asterisk ( * ).
  • B Multiple sequence alignment of yeast Sec2 and human Rabin8. Sequence homology is highlighted by arrows.
  • C Structural comparison of guanine effector binding region of Sec2 and Rabin8 by superposition of Ypt1 , Sec4 and Rab8A.
  • Rabin8 residues D203, E208, E210, E21 1 , E218 and E219 correspond to PDB residue numbering D187, E192, E194, E195, E202 and E203 (PDB code 4LHY; human crystal structure).
  • FIG 16 Schematic representation of PINK1 regulation of Rab8A Ser 111 phosphorylation.
  • PINK1 activation leads either to activation of a kinase or inhibition of a protein phosphatase that targets Ser 111 phosphorylation of Rab8A.
  • Phosphorylation of Rab8A at Ser 111 impairs Rabin8 (GEF) mediated GDP exchange leading to GDP-bound inactive Rab8A.
  • GEF Rabin8
  • Figure 17 Conservation of Ser 111 of Rab8A in 15 human Rab GTPases. Multiple sequence alignment of all human Rab GTPases in the region of Ser 111 of Rab8A reveals 15 Rabs with conservation of phosphorylation site for Ser or Thr. The phosphorylation residue is highlighted with an asterisk.
  • Tissue culture reagents were from Life Technologies. [ ⁇ - 32 ⁇ ] ATP was from Perkin Elmer. All mutagenesis was carried out using the QuikChange® site-directed- mutagenesis method (Stratagene) with KOD polymerase (Novagen). All DNA constructs were verified by DNA sequencing, which was performed by The Sequencing Service, School of Life Sciences, University of Dundee, using DYEnamic ET terminator chemistry (Amersham Biosciences) on Applied Biosystems automated DNA sequencers. DNA for mammalian cell transfection was amplified in E.coli DH5 strain and plasmid preparation was done using Qiagen Maxi prep Kit according to manufacturers' protocol. All cDNA plasmids and antibodies generated for this study are available to request from MRC-PPU Reagents (University of Dundee). All other reagents and chemicals were standard grade from Sigma or as indicated.
  • anti-Rab8A phosphor-Ser 111 S503D, 4 th bleed; raise against residues 104- 117 of human Rab8A: RNIEEHApSADVEKMR), anti-Rab8B phosphor-Ser 111 (S504D, 5 th bleed; raise against residues 104-1 17 of human Rab8B: RNIEEHApSSDVERMR), and anti-Rab13 phospho-Ser 111 (S505D, 8 th bleed; raise against residues 104-1 17 of human Rab13: KSIKENApSAGVERLR); anti-total PINK1 (for immunoprecipitation) (S774C, 3 rd bleed; raised against residues 235-end of mouse PINK1 ); anti-total PINK1 (for immunoprecipitation-imm
  • the mouse monoclonal anti-PINK1 antibody (human PINK1 residues 125-539) was raised by Dundee Cell Products.
  • Anti-Rab8A (for immunoprecipitation) and anti-GAPDH antibodies were obtained from Cell Signaling Technology.
  • Anti-Rab8 (for immunoblotting) and anti-HA agarose bead were obtained from Sigma. GFP binder sepharose beads were generated by the DSTT.
  • Anti- Parkin mouse monoclonal antibody was obtained from Santa Cruz.
  • Anti-CISD1 and anti-Rabin8 (Rab3IP) antibodies were obtained from Proteintech.
  • the rabbit monoclonal (NIAR164) anti-Mitofusin2 antibody was obtained from Abeam.
  • Anti-HA HRP antibody was obtained from Roche.
  • Anti-Parkin phosphor-Ser 65 rabbit monoclonal antibody was raised by Epitomics in collaboration with the Michael J Fox Foundation for Research.
  • Anti-LRRK2 and anti-LRKK2 phospho-Ser935 antibodies were obtained from Professor Dario Alessi (University of Dundee, UK).
  • Flp-ln T-Rex HEK293 cells expressing FLAG-empty, wild-type PINK1 -FLAG (WT), and kinase-inactive PINK1 -FLAG (Kl) were generated previously (Kondapalli, 2012).
  • CRISPR/Cas9 system generated PINK1 knockout (KO) HeLa cells were kindly provided by Richard Youle (NIH).
  • Flp-ln TRex HEK293 cells stably expressing GFP- LRRK2 were provided by Professor Dario Alessi (University of Dundee, UK) and have been described previously (Dzamko et al, 2010).
  • DMEM Dulbecco's modified Eagle's medium
  • FBS Fetal Bovine Serum
  • 2 mM L-Glutamine 100 U/ml penicillin and 0.1 mg/ml streptomycin at 37°C under a 5% C0 2 atmosphere.
  • MEF and HeLa cells were maintained using DMEM plus 1% (v/v) non-essential amino acid.
  • Flp-ln TRex HEK293 cells were maintained using DMEM plus 15 ⁇ g/ml of Blasticidin and 100 ⁇ g/ml of Hygromycin.
  • doxycycline was added to the medium for 24 hours.
  • Cell transfections were performed using polyethylenimine (Polysciences) or Lipofectamine 2000 (Life Technologies) according to manufacturer's instruction.
  • CCCP Carbonyl cyanide m- chlorophenylhydrazone
  • Fibroblasts were cultured in DMEM supplemented with glucose (4,5 g/l), L-Glutamine (2 mM), HEPES (10 mM), fetal bovine serum (10%), penicillin (50 U/ml)/streptomycin (50 ⁇ g/ml) plus 1% (v/v) non-essential amino acid, and grown at 37°C in a 5% C0 2 atmosphere.
  • MEFs Littermate matched wild type and homozygous PINK1 or Parkin knockout mouse embryonic fibroblasts (MEFs) were isolated from mouse embryos at day E13.5 resulting from crosses between heterozygous mice using a previously described protocol (Castor et al, 2013). Briefly, on day E13.5, the heads were used for genotyping. The red organs were removed and the embryo was minced and resuspended in 1 ml trypsin and incubated at 37°C for 15 min before the addition of 10 ml growth medium. Cells were plated and allowed to attach overnight before cells were washed with fresh medium to remove debris.
  • MEF cells were immortalised using SV40 large T antigen. All animal studies and breeding was approved by the University of Dundee ethical committee and performed under a U.K. Home Office project license.
  • Complementary oligonucleotides were designed and annealed to yield dsDNA inserts with compatible overhangs to Bbsl-digested vectors (Cong et al, 2013), the antisense guide was cloned into the spCas9 D10A expressing vector pX335 (Addgene Plasmid #42335) and the sense guide into the puromycin selectable plasmid pBABED P U6 (University of Dundee). HeLa cells were co-transfected with 1 ⁇ g of each plasmid using PEI in a 10 cM dish.
  • the PGR products were subcloned into pSC-B-amp/kan Vector using StrataClone Blunt PCR Cloning Kit (Agilent Technologies). Twelve positive clones (white colonies) were amplified and plasmid DNA was isolated, followed by restriction enzyme digestion analysis. The cloning of genomic PCR fragments allows for the isolation and sequencing of the single DNA molecules corresponding to each allele. Sequencing of the genomic PCR fragments from the knockout lines revealed a 1 10 base pair deletion (including start codon) and 70 base pair insertion (34+36 base pair insertions) and confirmed the presence of frameshifting indeis in region surrounding the ATG start codon of RAB8A.
  • Flp-ln T-Rex HEK293 cells expressing FLAG-empty, wild-type PINK1 -FLAG (WT), and kinase-inactive PINK1 -FLAG (Kl) were grown in 'light' (K0R0), 'medium' (K4R6) and 'heavy' (K8, R10) SILAC media, respectively, for at least 5 passages.
  • Cells in each condition were stimulated with 10 ⁇ CCCP for 3 hours and were scraped in appropriate amount of homogenization buffer (8.55 % w/v sucrose in 3 mM imidazole pH 7.4, supplemented with protease inhibitor and phosphatase inhibitor cocktail from Roche and benzonase from Roche).
  • the cells were lysed by mechanical disruption using a stainless steel homogenizer, unbroken cells and nuclei were removed by centrifugation at 1000 g for 10 minutes at 4°C.
  • the membrane fraction in the remaining post-nuclear supernatant was enriched by ultra-centrifugation and 100,000 g for 30 minutes (4°C).
  • RapiGest After alkylation with 5 mM iodoacetamide and subsequent quenching with 10 mM DTT, solutions were diluted to 0.1 % RapiGest using 50 mM Tris-HCI pH 8.0 and proteins were digested by Trypsin (1 :50) over night at 37 °C. RapiGest was cleaved by addition of 1 % trifluoroacetic acid (TFA) and removed by solid-phase extraction.
  • TFA trifluoroacetic acid
  • Normalised collision energy was set to 35, activation time was 10 ms.
  • Four hour linear gradients were performed from 5% solvent B to 35% solvent B (solvent A: 0.1 % formic acid, solvent B: 80% acetonitrile 0.08% formic acid) at 300 nl/min in 217 minutes with a 23 minute washing and re-equilibration step.
  • Protein identification and quantification were made using MaxQuant (Cox & Mann, 2008) Version 1 .3.0.5 with the following parameters: FT mass tolerance 20 ppm; MS/MS ion trap tolerance 0.5 Da; Trypsin/P set as enzyme; stable modification carbamidomethyl (C); variable modifications Oxidation (M), Acetyl (Protein N-term), Phospho (STY); maximum 5 modifications per peptide, and 2 missed cleavages. Searches were conducted using a combined Uniprot-Trembl Homo sapiens database with isoforms downloaded February 15, 2012 plus common contaminants (1 17,706 sequences). Identifications were filtered at a 1 % FDR at the peptide level, accepting a minimum peptide length of 7.
  • the 5 most intense ions, above a specified minimum signal threshold (5,000), based upon a low resolution (R 15,000) preview of the survey scan, were fragmented by collision induced dissociation and recorded in the linear ion trap, (Full AGC Target; 30,000. MSn AGC Target; 5,000).
  • Multi-Stage- Activation was used to provide a pseudo MS3 scan of any parent ions showing a neutral loss of 48.9885, 32.6570, 24.4942, allowing for 2+, 3+ and 4+ ions respectively.
  • Protein lysates were extracted in lysis buffer containing Buffers 50 imM Tris-HCI (pH 7.5), 1 mM EDTA, 1 mM EGTA, 1 % (w/v) Triton, 1 mM sodium orthovanadate, 10 mM sodium glycerophosphate, 50 mM sodium fluoride, 10 mM sodium pyrophosphate, 0.25 M sucrose, 0.1 % (v/v) 2-mercaptoethanol, 1 mM benzamidine, 0.1 mM PMSF and protease inhibitor cocktail (Roche). Lysates were clarified by centrifugation at 13,000 r.p.m. for 15 minutes at 4°C and the supernatant was collected. Protein concentration was determined using the Bradford method (Thermo Scientific) with BSA as the standard.
  • HA-tag Rab proteins For immunoprecipitation of HA-tag Rab proteins, 0.25-1 mg of protein extracts was undertaken by standard methods with anti-HA agarose beads. For immunoprecipitation of endogenous Rab8A cell lysates containing 1 mg of protein were immunoprecipitated at 4°C for at least 2 hours with 2 ⁇ of anti-Rab8A antibody pre-bound to 15 ⁇ of Protein A-agarose beads. The immunoprecipitates were washed three times with lysis buffer containing 0.15 M NaCI and eluted by resuspending in 20 ⁇ of 1 x SDS sample buffer.
  • Immunoprecipitates or cell extracts (25-50 ⁇ g of protein) were subjected to SDS/PAGE (4-12%) and transferred on to nitrocellulose membranes. Membranes were blocked for 1 hour in Tris-buffered saline with 0.1 % Tween (TBST) containing 5% (w/v) BSA. Membranes were probed with the indicated antibodies in TBST containing 5% (w/v) BSA overnight at 4°C. Detection was performed using appropriate HRP-conjugated secondary antibodies and enhanced chemiluminescence reagent.
  • Mitochondrial proteins were enriched as described previously (Kazlauskaite et al, 2015).
  • 200 ⁇ g of mitochondrial protein extracts was used for pull down with HALO-UBA UBQLN1 resin as described previously (Kazlauskaite et al, 2015).
  • yeast Ypt1 , yeast Sec4 and human Rab8A Multiple sequence alignment of yeast Ypt1 , yeast Sec4 and human Rab8A was made with T-COFFEE (default settings, v8.99). Alignment of the yeast Sec2 and human Rabin8 sequences was performed with Jalview's pairwise alignment function. Alignment of PDB structures containing Ypt1 , Sec4 and Rab8A was made with UCSF Chimera's matchmaker function (v 1 .10.1 with default parameters). Kinase assays and phosphorylation site mapping
  • recombinant Rab8A 24 ⁇ g was incubated with MBP-fused WT TcPINKI (50 ⁇ g) for 120 minutes in the same condition as the kinase assay, except [ ⁇ - 32 ⁇ ] ATP was approx. 20 000 cpm/pmol.
  • the reaction was terminated by addition of SDS sample buffer with 10 mM DTT, boiled and subsequently alkylated with 50 mM iodoacetamide before samples were subjected to electrophoresis on a Bis-Tris 4-12% polyacrylamide gel, which was then stained with Colloidal Coomassie blue (Invitrogen).
  • Protein bands were excised from the gel and 98% of the 32 P radioactivity incorporated into Rab8A was recovered from the gel bands after tryptic digestion.
  • Peptides were chromatographed on a reverse-phase HPLC Vydac C18 column (catalogue number 218TP5215, Separations Group) equilibrated in 0.1 % trifluoroacetic acid, and the column developed with a linear acetonitrile gradient at a flow rate of 0.2 ml/min before fractions (0.1 ml each) were collected and analysed for 32 P radioactivity by Cerenkov counting.
  • Isolated phosphopeptides were analysed by LC-MS/MS on a Thermo U3000 RSLC nano-LC system coupled to a Thermo LTQ- Orbitrap Velos Pro mass spectrometer.
  • the resultant data files were searched using Mascot (www.matrixscience.com) run on an in-house system against a database containing the Rab8A sequence, with a 10 p.p.m. mass accuracy for precursor ions, a 0.6 Da tolerance for fragment ions, and allowing for Phospho (S/T), Phospho (Y), Oxidation (M) and Dioxidation (M) as variable modifications.
  • Individual MS/MS spectra were inspected using Xcalibur v2.2 software (Thermo Scientific). The site of phosphorylation of these 32 P-labelled peptides was determined by solid-phase Edman degradation on a Shimadzu PPSQ33A sequencer of the peptide coupled to Sequelon- AA membrane (Applied Biosystems).
  • the wild-type (WT) or kinase-inactive Tribolium castaneum PINK1 (TcPINKI ) were expressed Escherichia coli and purified as described previously (Woodroof et al, 201 1 ).
  • the Rab8A WT was expressed in Escherichia coli BL21 (DE3) and purified as described previously (Bleimling et al, 2009).
  • the S1 1 1 E and S1 1 1A substitutions of Rab8A were introduced by site directed mutagenesis (QuikChange, Agilent Technologies, Santa Clara, CA, USA) and proteins were expressed and purified analogously to Rab8A WT.
  • OCRL1 (15 ⁇ ) and individual Rab proteins (19.5 ⁇ ) were incubated for 1 hour in a volume of 70 ⁇ _ and subjected to chromatographic separation on a Superdex 200 (10/30) gel filtration column (GE Healthcare, USA) using a HPLC system (Shimadzu, Japan) equipped with a SPD-20AV UV/Vis detector and detected at 254 nm.
  • the column was pre-equilibrated with 20 mM HEPES pH 7.5, 50 mM NaCI, 2 mM DTE, 1 mM MgCI 2 , 1 ⁇ GppNHp.
  • HORIBA Jobin Yvon Fluoromax-4 fluorescence spectrometer
  • the Rab-proteins (1 ⁇ ) were incubated with 100 ⁇ GDP in 1 ml_ buffer (20 mM HEPES pH 7.5, 50 mM NaCI, 1 mM MgCI 2 , 2 mM DTE) in a Quartz SUPRASIL cuvette (Hellma Analytics, Germany) and the reaction was started by addition of 0.5 ⁇ Rabin8. The decrease in mant fluorescence was used as a measure of mantGDP release.
  • the thermal shift assay can be used to investigate the stability of proteins (Ericsson et al, 2006).
  • the melting point of the protein is determined by the fluorescence of a dye (Sypro Orange, Sigma-Aldrich, USA).
  • the fluorescence of the dye is quenched in solution but remains when the dye is bound to hydrophobic regions. Through a successive increase in temperature, the protein unfolds and exposes more hydrophobic regions that the dye can bind to. This leads to an increase in fluorescence.
  • the assay was performed with 1 and 10 ⁇ g of the Rab proteins, respectively.
  • the proteins were mixed in a 1 :1 ratio with a 10x Sypro Orange solution in a total volume of 20 ⁇ _.
  • the probes were prepared as triplicates and the assay was performed in a 96- well plate in a RT PCR-cycler (Agilent Technologies Stratagene Mx3000P).
  • the Rab proteins (1 mg) were loaded with GTP by incubation with a 20 fold excess of GTP and 5 mM EDTA for 2 hours at RT. After the loading, excess GTP was removed by applying the protein solution to a Nap10 column (GE Healthcare, USA) and subsequent washing with buffer (20 mM HEPES pH 7.5, 50 mM NaCI, 1 ⁇ GTP, 2 mM DTE) according to the manufacturers manual. The analysis of intrinsic GTP hydrolysis was performed with 50 ⁇ protein. At defined time points, 20 ⁇ _ of the protein solution was denatured by incubation for 10 minutes at 95 °C and subsequently centrifuged to separate the protein and the nucleotide.
  • Rab8 proteins (1 mg) were loaded with GTP by incubation with a 20 fold excess of GTP and 5 mM EDTA for 2 hours at RT. After the loading, excess GTP was removed by applying the protein solution to Nap10 columns (GE Healthcare, USA) and subsequent washing with buffer (20 mM HEPES pH 7.5, 50 mM NaCI, 1 ⁇ GTP, 2 mM DTE) according to the manufactures manual. The analysis of the GAP-stimulated GTP-hydrolysis was performed with 30 ⁇ of the respective Rab8-variant and 100 nM of TBC1 D20.
  • SILAC-based PINK1 phosphoproteomic screen The present inventors, amongst others, have previously reported that the Parkinson's associated PINK1 kinase becomes activated in mammalian cells upon mitochondrial depolarization that can be induced by mitochondrial uncouplers such as CCCP (Kondapalli et al, 2012; Okatsu et al, 2012). This leads to phosphorylation of its substrates Parkin and ubiquitin at the equivalent residue Ser 65 (Kane et al, 2014; Kazlauskaite et al, 2014; Kondapalli et al, 2012; Koyano et al, 2014; Ordureau et al, 2014). To identify novel PINK1 -dependent phosphorylation targets the present inventors undertook a quantitative phosphoproteomic screen using stable-isotope labelling by amino acids in cell culture (SI LAC).
  • PINK1 activation is essential for Rab8A, Rab8B and Rab13 Ser 111 phosphorylation in cells
  • the present inventors next investigated whether endogenous PINK1 is sufficient and necessary for phosphorylation of Rab8A, Rab8B and Rab13 Ser 111 in cells upon activation induced by CCCP-induced mitochondrial depolarization. Wild-type HA- Rab8A, Rab8B and Rab13 as well as a non-phosphorylatable S1 1 1A mutants of each Rab GTPase were expressed in both wild-type and PINK1 knock-out HeLa cells generated by CRISPR/Cas9 technology (Narendra et al, 2013).
  • the inventors next analysed primary human fibroblasts derived from a patient with PD bearing the homozygous Q456X mutation and an unaffected individual from the same family. Using recombinant insect PINK1 in vitro kinase assays, the inventors have previously demonstrated that the Q456X mutation completely abolishes the catalytic activity of PINK1 via truncation of the C-terminal region that is essential for kinase function (Woodruff et al, 201 1 ).
  • Rab8A is not required for PI NK1 -dependent activation of Parkin E3 ligase activity
  • the inventors further confirmed this in MEFs derived from a Parkin knockout mouse model (Itier et al, 2003). Immunoprecipitation-immunoblot analysis revealed Rab8A Ser 111 phosphorylation in both wildtype and Parkin knockout MEFs after stimulation with CCCP (data not shown).
  • the inventors therefore undertook a comparative analysis of structural data available on the location of the phosphorylatable residues: ubiquitin Ser 65 , Parkin Ser 65 , Rab8A Ser 111 and the paralogous RabIA Ser 114 . Inspection of their structural environment ( Figure 10A) demonstrates that the phosphorylated sites in ubiquitin and Parkin have a markedly different structural environment to those of Rab 8A and 1A. In both ubiquitin and Parkin, the phosphorylated serine lies after a right-handed ⁇ -turn, before the 5th ⁇ - strand of the EF hand domain.
  • Rab GTPases belong to the superfamily of Ras GTPases and function as molecular switches cycling between GDP-bound inactive and GTP-bound active states (Hutagalung & Novick, 201 1 ).
  • GEFs guanine nucleotide exchange factors
  • GEFs physiologically catalyse the release of GDP thereby allowing Rab-activation by binding of GTP, which enables interaction with effector proteins that bind with high affinity to Rabs in their GTP-bound but not GDP-bound state.
  • the present inventors have previously structurally defined the interactions of Rab8A with its GEF Rabin8 (Guo et al, 2013).
  • Rabin8 is a 460 amino acid protein that contains a central Sec2 coiled-coiled domain exhibiting GEF activity towards Rab8 (Hattula et al, 2002). Whilst inspection of the co-crystal structure of Rab8A and Rabin8 revealed that Ser 111 is not directly involved in the formation of the interface of Rab8A and Rabin8, the side chain of Ser 111 lies close to a negative surface patch of Rabin8 adjacent to the interaction interface ( Figure 12A). The present inventors therefore hypothesised that addition of a negative charge on Ser 111 may influence the Rab8A-Rabin8 interaction.
  • the inventors preparatively loaded Rab8A with the fluorescent GDP analogue mantGDP and monitored the Rabin8-catalyzed time-dependent displacement of mantGDP in the presence of excess GDP as judged by the decrease of Mant- fluorescence ( Figure 12B).
  • GAPs GTPase activating proteins
  • the inventors next addressed whether phosphorylation of Rab8A at Ser 111 influenced the interaction of Rab8A and Rabin8 in cells.
  • the inventors expressed wildtype (WT) HA-Rab8A, a phosphomimetic S1 1 1 E mutant, and a S1 1 1 A mutant of HA-Rab8A in HeLa Rab8A knockout cells. Lysates were subjected to immunoprecipitation using HA- agarose following by immunoblotting of immunoprecipitates with anti-Rabin8 antibody and the inventors observed co-immunoprecipitation of endogenous Rabin8 with WT HA-Rab8A ( Figure 14A).
  • the negative surface patch of Rabin8 adjacent to the Rab8A interaction interface is comprised of residues Asp187 (D187), Glu192 (E192), Glu194 (E194) and Glu 195 (E195).
  • D187 residues Asp187
  • E192 Glu192
  • E194 E194
  • E195 Glu 195
  • yeast lacks PINK1 The inventors first verified that yeast lacks PINK1 . Examination of the entry for PINK1 in the EggNOG (Powell et al, 2014) orthologue database suggests PINK1 is only found in metazoans. We also employed the EggNOG hidden markov model for PINK1 to search the NCBI NR protein sequence database with the EMBL-EBI HMMER3 server. No significant matches were found in Saccharomyces (data not shown). Next, Rab8 was compared to Ypt1 and Sec4, its close homologues in yeast. Multiple sequence alignment of Ypt1 , Sec4 and Rab8A showed that whilst these sequences align well, Rab8A Ser 111 is neither conserved in Ypt1 nor Sec4 (data not shown).
  • the loop containing Ser 111 in Rab8A is not similar to that of Sec4, but in Ypt1 the corresponding residue is a threonine, followed by a serine.
  • Structural data for Ypt1 and Rab8A demonstrates that they exhibit a high degree of structural homology, and it is probable that Ser1 12 (Ser 112 ) of Ypt1 would be targeted in a PINK1 independent manner (Figure 15A).
  • Rab GTPases cycle between active GTP-bound and inactive GDP bound state that differ mainly by the conformation of two guanine nucleotide binding loops known as Switch I and Switch II regions (Hutagalung & Novick, 201 1 ).
  • Switch I and Switch II regions Hutagalung & Novick, 201 1 .
  • the Rab8-Rabin8 complex there is a direct interaction of the Switch II region with the GEF that is a universal feature of all currently known GTPase-GEF complexes (Guo et al, 2013).
  • There are additional sites of interaction of the Switch I region with Rabin8 that have are also been reported for other GTPase-GEF complexes (Guo et al, 2013).
  • PINK1 dependent pathways are linked to pathways mediated by other PD-linked genes.
  • Pathologically PD is defined by the presence of cytoplasmic inclusions known as Lewy bodies whose major protein component is a-synuclein.
  • Post-mortem analysis of brains from a family with Parkinsonism harbouring PINK1 mutations has confirmed the presence of Lewy bodies in the substantia nigra (Samaranch et al, 2010).
  • mutations post-mortem analysis of brains from a family with Parkinsonism harbouring PINK1 mutations has confirmed the presence of Lewy bodies in the substantia nigra (Samaranch et al, 2010).
  • mutations in the substantia nigra
  • Hutagalung AH, Novick PJ (201 1 ) Role of Rab GTPases in membrane traffic and cell physiology.
  • Itier JM, Ibanez P Mena MA, Abbas N, Cohen-Salmon C, Bohme GA, Laville M, Pratt J, Corti O, Pradier L, Ret G, Joubert C, Periquet M, Araujo F, Negroni J, Casarejos MJ, Canals S, Solano R, Serrano A, Gallego E, Sanchez M, Denefle P, Benavides J, Tremp G, Rooney TA, Brice A, Garcia de Yebenes J (2003) Parkin gene inactivation alters behaviour and dopamine neurotransmission in the mouse. Human molecular genetics 12: 2277-2291
  • PINK1 is activated by mitochondrial membrane potential depolarization and stimulates Parkin E3 ligase activity by phosphorylating Serine 65.

Abstract

La présente invention concerne un biomarqueur pour la maladie de Parkinson. Le biomarqueur et des produits associés au biomarqueur peuvent être utilisés afin d'aider au diagnostic ou d'évaluer l'apparition et/ou le développement de la maladie de Parkinson. L'invention concerne également l'utilisation du biomarqueur dans l'évaluation de traitements médicamenteux, l'analyse de médicaments ou le développement de médicaments dans le domaine de la maladie de Parkinson et des troubles associés à la maladie de Parkinson.
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