Given the observed effects of LRRK2 kinase activity on synaptic vesicle trafficking, we decided to explore potential LRRK2 presynaptic substrates. A number of putative presynaptic proteins have been previously identified to be able to interact with LRRK2 (Piccoli et al., 2014). We chose to explore the relationship between LRRK2 and NSF, an AAA+ ATPase, since LRRK2 kinase activity appears involved in the control of a step of the exo-endocytosis (paragraph 7.1) and NSF plays a pivotal role in SNARE complex disassembly and, hence, SV endocytosis (Zhao and Brunger, 2015). Moreover, NSF activity is tightly controlled by phosphorylation (Liu et al., 2006; Matveeva et al., 2001) and the ATPase has been found to accumulate in neuronal intranuclear inclusion bodies,
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similar to Lewy Bodies, a pathological hallmark of PD (Pountney et al., 2008). Thus, to assess whether NSF is a LRRK2 substrate we first set out to find the optimal conditions to purify human recombinant NSF.
As reported in the introduction, NSF belongs to the AAA+ (ATPases Associated with various cellular Activities) superfamily of proteins and it is responsible of the SNARE complex disassembly during vesicle recycling. Until now structural studies have been focused on recombinant Chinese Hamster (Cricetulus Griseus) NSF purified from E. coli (Chang et al., 2012; Zhao et al., 2015), whilst no studies are available on human NSF. Our aim was to first purify human NSF from mammalian HEK293T cells and then characterize it biochemically. Although the sequence alignment of C. Griseus and Human NSF reveals an identity of 98% between the two proteins (Fig.7.2.1), we reasoned that expressing and purifying recombinant NSF from mammalian cells would be important in terms of post-translational modifications that E. coli cannot provide.
70 SQVAFEKAENSSLNLIGKAKTKENRQSIINPDWNFEKMGIGGLDKEFSDIFRRAFASRVF CrG_NSF 181 SQVAFEKAENSSLNLIGKAKTKENRQSIINPDWNFEKMGIGGLDKEFSDIFRRAFASRVF 240 Hum_NSF 241 PPEIVEQMGCKHVKGILLYGPPGCGKTLLARQIGKMLNAREPKVVNGPEILNKYVGESEA 300 PPEIVEQMGCKHVKGILLYGPPGCGKTLLARQIGKMLNAREPKVVNGPEILNKYVGESEA CrG_NSF 241 PPEIVEQMGCKHVKGILLYGPPGCGKTLLARQIGKMLNAREPKVVNGPEILNKYVGESEA 300 Hum_NSF 301 NIRKLFADAEEEQRRLGANSGLHIIIFDEIDAICKQRGSMAGSTGVHDTVVNQLLSKIDG 360 NIRKLFADAEEEQRRLGANSGLHIIIFDEIDAICKQRGSMAGSTGVHDTVVNQLLSKIDG CrG_NSF 301 NIRKLFADAEEEQRRLGANSGLHIIIFDEIDAICKQRGSMAGSTGVHDTVVNQLLSKIDG 360 Hum_NSF 361 VEQLNNILVIGMTNRPDLIDEALLRPGRLEVKMEIGLPDEKGRLQILHIHTARMRGHQLL 420 VEQLNNILVIGMTNRPDLIDEALLRPGRLEVKMEIGLPDEKGRLQILHIHTARMRGHQLL CrG_NSF 361 VEQLNNILVIGMTNRPDLIDEALLRPGRLEVKMEIGLPDEKGRLQILHIHTARMRGHQLL 420 Hum_NSF 421 SADVDIKELAVETKNFSGAELEGLVRAAQSTAMNRHIKASTKVEVDMEKAESLQVTRGDF 480 SADVDIKELAVETKNFSGAELEGLVRAAQSTAMNRHIKASTKVEVDMEKAESLQVTRGDF CrG_NSF 421 SADVDIKELAVETKNFSGAELEGLVRAAQSTAMNRHIKASTKVEVDMEKAESLQVTRGDF 480 Hum_NSF 481 LASLENDIKPAFGTNQEDYASYIMNGIIKWGDPVTRVLDDGELLVQQTKNSDRTPLVSVL 540 LASLENDIKPAFGTNQEDYASYIMNGIIKWGDPVTRVLDDGELLVQQTKNSDRTPLVSVL CrG_NSF 481 LASLENDIKPAFGTNQEDYASYIMNGIIKWGDPVTRVLDDGELLVQQTKNSDRTPLVSVL 540 Hum_NSF 541 LEGPPHSGKTALAAKIAEESNFPFIKICSPDKMIGFSETAKCQAMKKIFDDAYKSQLSCV 600 LEGPPHSGKTALAAKIAEESNFPFIKICSPDKMIGFSETAKCQAMKKIFDDAYKSQLSCV CrG_NSF 541 LEGPPHSGKTALAAKIAEESNFPFIKICSPDKMIGFSETAKCQAMKKIFDDAYKSQLSCV 600 Hum_NSF 601 VVDDIERLLDYVPIGPRFSNLVLQALLVLLKKAPPQGRKLLIIGTTSRKDVLQEMEMLNA 660 VVDDIERLLDYVPIGPRFSNLVLQALLVLLKKAPPQGRKLLIIGTTSRKDVLQEMEMLNA CrG_NSF 601 VVDDIERLLDYVPIGPRFSNLVLQALLVLLKKAPPQGRKLLIIGTTSRKDVLQEMEMLNA 660 Hum_NSF 661 FSTTIHVPNIATGEQLLEALELLGNFKDKERTTIAQQVKGKKVWIGIKKLLMLIEMSLQM 720 FSTTIHVPNIATGEQLLEALELLGNFKDKERTTIAQQVKGKKVWIGIKKLLMLIEMSLQM CrG_NSF 661 FSTTIHVPNIATGEQLLEALELLGNFKDKERTTIAQQVKGKKVWIGIKKLLMLIEMSLQM 720 Hum_NSF 721 DPEYRVRKFLALLREEGASPLDFD 744 DPEYRVRKFLALLREEGASPLDFD CrG_NSF 721 DPEYRVRKFLALLREEGASPLDFD 744
Fig.7.2.1 | Protein blast alignment: The result of full-length sequence alignment between
Human NSF (Hum_NSF) and Cricetulus Griseus NSF (CrG_NSF) displays an identity on 732/744 residues (98%) among the three different domain (N-terminal 1-205, yellow; D1 206-487, red; D2 488-744, green).
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We first validated recombinant human Flag-tagged NSF full-length, purified according to the procedures described in materials and methods. The flag tag is a short amino-acid peptide (DYKDDDDK), fused to human NSF at the N-terminal, widely used in a variety of cell types, including bacteria, yeast and mammalian cells. Furthermore, flag-tagged proteins have the advantage that can be purified by immunoprecipitation from the cell lysate using beads conjugated with antibodies and then eluted from the resin by competition with an excess of Flag peptides. Starting from 20x106 cells, we obtained
about 60 µg of highly pure monomeric human NSF at a concentration of 0.3 mg/ml (Fig.7.2.2 A). Since SDS-PAGE gel electrophoresis allows to detect only denatured NSF due to the presence of both sodium dodecyl sulfate (SDS) and dithiotreitol (DTT), we subsequently monitored the ability of purified proteins to assemble into hexamers. A common feature of all the AAA+ proteins is the Walker A motif, whose function is necessary for ATP binding and conformational changes (Hanson and Whiteheart, 2005). For this reason, to assess the capability of NSF to undergo from monomeric to homoexameric structure, we incubated the purified protein with 1 mM ATP and 4 mM MgCl2. Based on the Cryo-EM structure of Chinese Hamster NSF recently published
(Chang et al., 2012 e mechanicistic), we monitored the presence of hexamers in solution by Transmission Electron Microscopy (TEM). As shown in Fig.7.2.2 B, the presence of a structural transition into oligomeric state denotes the solubility of the protein and confirms that human flag-tagged NSF is able to form hexamers in the presence of ATP.
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SDS-PAGE and quantified by comparison with different concentration of BSA (Bovine Serum Albumin); (B) (A) Purified human NSF separated on SDS-PAGE and quantified by comparison with different concentration of BSA (Bovine Serum Albumin); (B) Representative TEM micrograph of purified NSF with 1 mM ATP and 4 mM MgCl2 reveals the presence of hexamers as highlighted in
the large onset. (scale bar 50 nm).
In addition, we exploited size exclusion chromatography (SEC) to further monitor the ability of flag-tagged NSF to oligomerize upon ATP addition. To this aim, different amounts of ATP were added to the cell lysates and NSF-containing lysates were subsequently loaded into a Superdex200 gel filtration column (Fig.7.2.3). The elution profile of NSF was then monitored by a dot blot analysis collecting all the chromatographic fractions. As shown in Fig.7.2.3, the addition of the nucleotide causes a shift in the position of NSF elution peak toward shorter retention times, suggesting an increased hydrodynamic radius of the protein in presence of ATP. Moreover, the entity of the shift of the NSF chromatographic peak is different as a function the ATP concentration. The presence of an intermediate peak at 90 µM of ATP between 0 and 1 mM suggests that NSF may assemble into intermediate species before acquiring its hexameric conformation. Supporting this finding, early studies indicate that NSF can form trimers and that the two ATP binding sites (D1 and D2) possess different affinities for the nucleotide (Whiteheart et al., 1994; Matveeva et al., 1997).
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Fig.7.2.3 | SEC profile of cell lysates overexpressing flag-NSF adding different ATP concentration: Size exclusion chromatography fractions of HEK293T expressing flagged-NSF
spotted onto nitrocellulose membrane and probed with anti-flag antibody; SEC profile of cell lysates adding different ATP concentrations in the Lysis buffer.
Interestingly, Flag-NSF purified with M2-Flag agarose beads binds endogenous NSF, as indicated by the presence of an extra band with an approximate molecular weight of the endogenous protein (82 kDa) and that is recognized by an antibody against NSF but not by the anti-flag antibody (Fig.7.2.4).
Fig.7.2.4 | Co-purification of endogenous NSF: western-blot analysis to validate the presence of
endogenous NSF co-purification with recombinant human NSF using anti-flag and anti-NSF antibodies.
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form its functional hexameric structure, the next step was to evaluate NSF ATPase activity. We set up two complementary assays to monitor the catalytic activity of NSF: 1) a Malachite Green Enzyme assay and 2) an isocratic reversed-phase HPLC (RP-HPLC)- based assay. The latter, provides a direct measurements of ATP to ADP conversion by monitoring the areas of the peaks corresponding to the two nucleotides. The Malachite Green is a colorimetric assay that measures the inorganic phosphate (Pi) free in solution, released from ATP during the reaction (Lanzetta et al., 1979). Thus, it is an indirect measurement influenced by several aspects, such as Pi already present in the reaction buffer and/or produced by other sources (i.e. contaminating GTPases/ATPases). However, this assay has the advantage of being faster and scalable, since it measures the absorbance (at a wavelength of 640 nm) of the reaction at a given time (Fig.7.3.1).
Fig.7.3.1 | Pi free in solution measured with Malachite Green assay: Schematic
representation of the Malachite Green enzyme assay, on top, and the concentration of Pi free in solution as a function of time ([ATP] = 0.5 mM). Aliquots were collected as a function of time and subtracted by a control purification of untransfected cells. Velocities of the reactions (slopes) were calculated by linear regression using GraphPad Prism 5 software.
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To validate the Malachite Green assay, we performed it in parallel with the HPLC-based assay, which provides a direct measurement of the ATP consumption and concomitant ADP formation, but has the limitation of requiring long running times and it cannot be implemented in a high-throughput fashion. For the Malachite Green assay, we collected aliquots of the reaction at different time points and evaluated the Pi formation by measuring the absorbance at 640 nm, as show in Fig.7.3.1. For the RP-HPLC assay, we detected the variation of ATP and ADP over time by injecting aliquots of the reaction collected at different time points (Fig.7.3.2). Both assays confirmed that human flag-tag NSF purified from mammalian cells is an active ATPase: as shown in Fig.7.3.2 B and 7.3.1, the amount of ADP and the concentration of Pi in solution increased as a function of the reaction time.
Fig.7.3.2 | Isocratic RP-HPLC assay: (A) Reaction profile of the two different nucleotide peaks
over time ([ATP] = 0.7 mM). The ATPase activity of NSF was evaluated measuring the area under the peak of ADP (B). Aliquots were collected as a function of time and subtracted a control purification of untransfected cells. Velocities of the reactions (slopes) were calculated by linear regression using GraphPad Prism 5 software.
We then identified the best purification conditions to maximize the ATPase activity of NSF in vitro. Specifically, we tested if the presence Tween-20 has a negative effect on NSF catalytic activity. For this reason, we purified NSF in parallel with i) lysis buffer containing 1% of detergent, a purification protocol routinely used to purify LRRK2, and ii) with repeated freeze/thaw cycles. This new tested protocol was used because it is a gentle process of lysing cells and it can help maintaining the protein folding. After having validated, with a BCA assay, that 5 cycles are enough to disrupt plasma membranes, we
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Fig.7.3.3 | Impact of Tween-20 on NSF ATPase activity: (A) Concentration of Pi free in solution with different amount of the substrate reveal that the rate of the reaction decrease in presence of the detergent, as highlight in (B) fitting slopes with Michaelis-Menten equation (NSF+Tween- 20 Vmax=1.24 µmol/min; NSF Vmax=2.32 µmol/min).
Based on these observations, all the subsequent NSF purifications were carried out using a freeze/thaw protocol and the ATPase activity of NSF was measured using the Malachite Green assay.
Next, we determined the KM of NSF using the Malachite Green enzyme assay. The KM
represents the concentration of substrate required to reach half of the reaction maximum velocity (Vmax) and provides an indication of the affinity of the substrate for
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the enzyme. We monitored this value by comparing proteins at different days post- purification at 4°C. We found that the value of the Km did not change, however the Vmax substantially decreased (Fig.7.3.4 B). This phenomenon suggests that a fraction of the protein progressively becomes inactive, but the fraction of active NSF has unaltered affinity for the substrate (Fig.7.3.4 A). Collecting all the data obtained from independent experiments with different amount of ATP, we showed that human NSF possesses a Km of 180±10 μM.
Fig.7.3.4 | Km of recombinant flag-NSF: The values of Km (A) and Vmax (B) calculated for NSF on
different days after purification, keeping the protein at 4°C. Km value was equal to 180±10 µM and did not change in function of days, suggesting that this corresponds to its affinity for the substrate.
Taken together, these observations indicate that recombinant human Flag-NSF full length purified from mammalian HEK293T cells is an active ATPase.