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Definitions

• the present disclosure relates to a method of performing an assay to monitor the overall autophagic flux within a host cell particularly, macroautophagy, pexophagy and other autophagic pathways.
• the present disclosure also relates to a vector or expression construct and host cell suitable for being employed in the assay to monitor autophagy and a kit thereof.
• Autophagy is the major intracellular catabolic pathway responsible for the turnover of cellular organelles and long-lived macromolecules. It regulates diverse biological events including development, aging, cell death and microbial infections. Autophagy and related pathways involve degradation of either selective or non- specific cytoplasmic components via double membrane vesicles. The pathway is initiated when a newly formed double membrane, the phagophore, expands and confines a part of the cytoplasm resulting in the formation of an autophagosome. The subsequent fusion of the autophagosome with a lysosome results in the formation of an auto lysosome and the degradation of the inner membrane and the cargo. This conserved pathway from yeast to humans plays critical role in cell survival during nutritional deprivation, clearance of damaged/superfluous organelles, protein aggregates and intracellular pathogens. Dysfunction of autophagy leads to cell death, cancer, neurodegenerative and other diseases.
• Autophagy helps cell survival especially during nutritional deprivation or starvation by recycling redundant, excess and/or superfluous cytoplasmic constituents including proteins and organelles.
• cytoplasmic constituents including proteins and organelles.
• pathogens including bacteria (Salmonella, Shigella, Mycobacterium, Group A Streptococcus, etc.) and viruses (herpes simplex virus, HIV, etc.) subvert autophagy to prevent their elimination by degradation in host lysosomal compartments.
• Autophagy also has a neuroprotective role, as it clears large aggregates of mutant polyubiquitylated proteins resistant to proteasomal degradation.
• autophagy has been shown to be involved in heart diseases, atherosclerosis, certain myopathies, innate and adaptive immune responses, Crohn's disease and cancer.
• targeting autophagy is a potentially exciting avenue that is just beginning to be exploited for disease and cancer cures.
• modulating autophagy has positive outcomes in the diseases mentioned above. For e.g., modulating autophagic activity either genetically or biochemically, have resulted in increased killing of intracellular mycobacteria.
• the instant invention aims at overcoming the above mentioned issues with a multiple assay which provides an effective alternative of a more robust, reproducible and sensitive kinetic assay to study pexophagy as well as general autophagy flux.
• the present disclosure relates to a method of performing an assay to monitor autophagy within a host cell, said method comprising acts of a) preparing at least one expression construct with a reporter gene b) transforming a host cell with the prepared expression construct c) culturing the transformed host cell in induction medium followed by starvation medium to induce autophagy within the host cell d) lysing the induced host cell of step (c) in a lysis buffer e) treating the lysed host cell with a substrate capable of reacting with the reporter to produce a light reaction, and f) analysing the reaction to monitor the autophagy; a vector or expression construct comprising a POT-1 promoter sequence and reporter gene optionally along with an SKL signal sequence; a host cell transformed with the vector or the expression construct as above; a method of identifying compound having or suspected of having an effect on autophagy, said method comprising act of a) performing steps a) to c) of the method as above to obtain the induced host cell; and
• Figure 1 shows plasmid constructs for expression of firefly and Renilla luciferase in S. cerevisiae (MTCC 5858- wild type cells of S.cerevisiae having only Renilla luciferase
• MTCC 5860- atg 1 mutant of S.cerevisiae having only firefly luciferase MTCC 5861- atg 1 mutant type having Renilla as well as firefly luciferase).
• Figure 1 (a) shows the pPM5 vector comprising the following:
• Vector Backbone - pRS305.Antibiotic Resistance - Ampicillin. Yeast Selection Marker -
• Renilla luciferase gene (Rluc) during peroxisome biogenesis.
• Figure 1 (b) shows the pPMlO vector comprising the following:
• Vector Backbone - pRS306.Antibiotic Resistance - Ampicillin. Yeast Selection Marker -
• Figure 2 (a) shows the pPM7 vector comprising the following:
• Vector Backbone - pJCF-214. Antibiotic Resistance - Ampicillin. Yeast Selection Marker - Arginine.
• Vector Description - Renilla luciferase gene downstream of Peroxisomal thiolase promoter of Pichia pastoris (Pp POT1) to drive the cytoplasmic expression of Renilla luciferase gene (Rluc) during peroxisome biogenesis.
• Figure 2 (b) shows the pPM9 vector comprising the following:
• Vector Backbone - pIBl Antibiotic Resistance - Ampicillin. Yeast Selection Marker - Histidine.
• Vector Description Flue gene with peroxisomal targetting sequence (SKL) cloned downstream of Pp POT1 promoter for peroxisomal expression of firefly luciferase gene.
• Figure 3 depicts a graph showing the activity of firefly and Renilla luciferase in wild type and mutant S. cerevisiae cells on induction of autophagy.
• Figure 3 a shows the dual luciferase assay in the wild type S. cerevisiae cells.
• Figure 3b shows the dual luciferase assay in autophagy mutant. As can be seen upon induction of autophagy the activity of luciferase goes down with time in case of wild type cells, whereas there is no decrease in the autophagy mutants.
• Figure 4 depicts a graph showing firefly luciferase assay in P. pastoris. The graph shows the decrease in firefly activity with time upon induction of autophagy. Luciferase assay is done in a 96 well plate with 3 independent assays done in triplicates and SD plotted as error bars.
• Figure 5 shows the selective degradation of a cargo marker indicative of selective autophagy (pexophagy) which is followed over time in the yeast Pichia pastoris.
• the rates of degradation of the untreated cells of Pichia Pastoris are compared to the ones treated with 50 ⁇ concentration of cyclic peptoids.
• the time taken for 50% decrease in cargo activity is taken as the criteria for comparing the control with the compounds ( Figure 5 a).
• the compounds that differ from the control by 3 SD (Standard Deviation) units are considered significant.
• the compound 4a shows significant difference from the control. It increases the rate of degradation of the protein marker through autophagy.
• a dose response assay for 4a shows a proportional increase in the rate of degradation with increasing concentration (Figure 5b).
• RLU % Relative Light Units
• Figure 7 shows a comparison between luciferase based assay and conventional Potl-GFP processing assay.
• Figure 7a shows the decrease in the activity of wild type cells with time and the constant activity exhibited by the mutant without any decrease, with time.
• Figure 7b shows a Western for luciferase protein levels.
• Figure 7c shows a Western for Pot-1 GFP processing assay.
• the present disclosure relates to a method of performing an assay to monitor autophagy within a host cell, said method comprising acts of:
• step (c) lysing the induced host cell of step (c) in a lysis buffer
• method of identifying compound having or suspected of having an effect on autophagy comprises acts of:
• the expression construct is prepared using vector backbone selected from a group comprising pRS305, pRS306, pIBl and pJCF-214 or any combination thereof.
• the reporter gene is selected from a group comprising firefly luciferase, Renilla luciferase and green fluorescent protein or any combination thereof.
• the expression construct comprises a ScPOT-1 promoter or PpPOT-1 promoter, optionally along with SKL signal sequence.
• the host cell is selected from a group comprising Saccharomyces cerevisiae and Pichia pastoris; and wherein the transforming of the host cell is carried out by lithium acetate method in Saccharomyces cerevisiae and by electroporation in Pichia pastoris.
• the induction medium is oleate medium comprising about 0.1% oleate, about 0.5% Tween-40, about 0.25%> yeast extract, about 0.5%> peptone and about 5mM phosphate buffer or wherein the induction medium is methanol medium comprising 1% (w/v) yeast extract, 2% (w/v) Bacto-peptone and 0.5%> methanol.
• the starvation medium is nitrogen starvation medium SD-N comprising, about 2% dextrose and about 0.17% yeast nitrogen base without amino acids and nitrogen source.
• the induction medium induces formation of peroxisomes in the host cell; and wherein the starvation medium induces autophagy, pexophagy, macro-autophagy or any combination thereof.
• the lysis buffer is passive lysis buffer having a pH ranging from about 6 to about 8, preferably 7.
• the substrate is luciferin which is added at a concentration ranging from about 8 ⁇ 1 to about 12 ⁇ , preferably 10 ⁇ .
• the autophagy is monitored by measuring decay of the light reaction with respect to time.
• said method is employed for identifying modulators of autophagy, in detecting mutants which are partially blocked or completely blocked in autophagy and in detecting degraders of peroxisomes.
• the present disclosure also relates to a vector or expression construct comprising a POT-1 promoter sequence and reporter gene optionally along with an SKL signal.
• the vector or expression construct is selected from a group comprising sequence set forth as SEQ ID Nos. 1, 2, 3 and 4.
• the reporter gene is selected from a group comprising firefly luciferase, Renilla luciferase and green fluorescent protein or any combination thereof.
• the POT-1 promoter is ScPOT-l promoter or pPOT-1 promoter.
• the present disclosure also relates to a host cell transformed with the vector or the expression construct as above.
• the host cell is selected from a group comprising Saccharomyces cerevisiae and Pichia pastoris.
• the present disclosure also relates to a method of identifying compound having or suspected of having an effect on autophagy, said method comprising act of: a. performing steps a) to c) of the method as above to obtain the induced host cell; and
• the compound is selected from a group comprising modulators of autophagy, mutants which are partially blocked or completely blocked in autophagy or degraders of peroxisomes.
• the present disclosure also relates to a kit to monitor autophagy, said kit comprising vector or expression construct or host cell as above, optionally along with components selected from a group comprising induction medium, starvation medium, lysis buffer, substrate and instruction manual or any combination thereof.
• the induction medium is oleate medium comprising about 0.1% oleate, about 0.5%> Tween-40, about 0.25%> yeast extract, about 0.5%> peptone and about 5mM phosphate buffer or wherein the induction medium is methanol medium comprising 1% (w/v) yeast extract, 2% (w/v) Bacto-peptone, 0.5%> methanol.
• the starvation medium is nitrogen starvation medium SD-N comprising, about 2% dextrose and about 0.17% yeast nitrogen base without amino acids and nitrogen source.
• the lysis buffer is passive lysis buffer having a pH ranging from about 6 to about 8, preferably 7.
• the substrate is luciferin which is added at a concentration ranging from about 8 ⁇ 1 to about 12 ⁇ , preferably 10 ⁇ .
• the autophagy comprises macro- autophagy, pexophagy, xenophagy and other related autophagic pathways or any combination thereof.
• the present disclosure relates to a dual luciferase based assay in yeast to monitor macroautophagy, pexophagy by a) creating plasmid constructs expressing firefly and Renilla luciferase b) generating yeast strains expressing these luciferase constructs, and c) developing autophagy assay using these strains.
• the said assay is carried out using live yeast cells with two built-in reporters for two different autophagy pathways (general and selective). Since the assay consists of two reporters which can differentiate and effectively measure two different forms of autophagy, the assay has been referred as 'dual'.
• pulse chase-like assay is employed, where the cargo, peroxisomal targeted firefly luciferase is built-up in peroxisomes and then its degradation is followed upon autophagy induction.
• the Renilla luciferase gene is without any targeting signal.
• Saccharomyces cerevisiae and Pichia pastoris are employed to study macro-autophagy, pexophagy, xenophagy and other autophagic related pathways, in particular pexophagy and macro-autophagy.
• peroxisomes in Saccharomyces cerevisiae are induced by growing the cells in oleate medium.
• peroxisomes in Pichia pastoris are induced by growing the cells in methanol medium.
• kinetic assay of pexophagy is used to detect mutants that are completely blocked or partially blocked in pexophagy. The degradation rates of all mutants tested are compared to the wild type. A mutant blocked completely in pexophagy would behave like the Aatgl mutant, showing no decrease in the luciferase activity with time. Other mutants such as the Aatg5 and Aatg8 mutants would also behave in the same way as Aatgl mutant as all these are core autophagy genes.
• a partial blocker can be identified by comparing the rates of degradation of luciferase reporter of the mutant with the wild type cells.
• the mutants, fast degraders that degrade peroxisomes much rapidly than the wild type are detected similarly, slow degraders are also detected.
• Saccharomyces cerevisiae shuttle vectors pRS306 (URA) and pRS305 (LEU) are used to clone the POT1 promoter and the firefly and Renilla luciferase genes, respectively.
• Pichia pastoris shuttle vectors pIBl (HIS) and pJCF-214 (ARG) are used to clone the POT1 promoter and the firefly and Renilla luciferase genes, respectively.
• the oleate responsive region of the POT1 promoter is amplified from yeast genomic DNA along with the firefly luciferase gene and Renilla luciferase gene.
• peroxisomal thiolase fused to GFP is used to monitor the pexophagy in yeast.
• Potl-GFP assay is based on the immunoblot analysis.
• autophagy assay provides the convenience of performing small molecule high through-put screening.
• the present disclosure relates to an assay to monitor autophagy.
• a vector or an expression construct is prepared, having a reporter gene and a promoter, optionally along with a signal sequence, wherein the promoter is a peroxisome specific promoter.
• the expression of such reporter gene is driven by said promoter, which is activated by induction of peroxisomes.
• This vector or expression construct is transformed into a host cell.
• plurality of vectors or expression constructs are inserted into a single host cell, to monitor autophagy, by single or multiple reporter systems.
• the host cell comprising single or multiple vectors or expression constructs is cultured in induction medium to induce formation of peroxisomes.
• This host is thereafter transferred and harvested on nitrogen starvation medium to induce autophagy, including pexophagy and macro-autophagy within the host cell.
• the host cell is thereafter subjected to lysis by employing a lysis buffer.
• a suitable substrate capable of reacting with the reporter to produce a light reaction is added. This light reaction is measured and analysed to monitor the autophagy, specifically by measuring decay of the light reaction with respect to time.
• the said assay is further employed to identify compounds having or suspected of having an effect on autophagy, by initially introducing the compounds in high throughput assay plate, followed by introducing the induced host cell into the assay plate. Thereafter similar steps of lysis and addition of substrate to observe the light reaction are performed. The change in decay of the emitted light, when compared to control conditions [without the test compound], will provide information on the effect of the test compounds on autophagy.
• the Pot 1 promoter is PCR amplified from S. cerevisiae genomic DNA.
• the amplicon is subjected to agarose gel electrophoresis against a suitable marker.
• the amplified 251 bp on the gel is eluted using Qiagen spin columns.
• the lacZ cassette from the vectors pRS306 (URA) is removed by digesting with Hindlll. Further, this digested vector is ligated with amplified POT1 promoter at the site of lacZ.
• the firefly luciferase gene with the peroxisomal targeting signal (SKL) is amplified by polymerase chain reaction from a vector such as plasmid pRSVL.
• the amplified firefly luciferase gene is ligated downstream of the POT1 promoter of pRS306 to obtain firefly luciferase construct (Vector I).
• the Pot 1 promoter is PCR amplified from Saccharomyces cerevisiae genomic DNA.
• the amplicon is subjected to agarose gel electrophoresis against a suitable marker.
• the amplified 25 lbp on the gel is eluted using Qiagen spin columns.
• the lacZ cassette from the vectors pRS305 (LEU) is removed by digesting with PvuII. Further, this digested vector is ligated with amplified POT1 promoter at the site of lacZ. Further, the Renilla luciferase gene is amplified by polymerase chain reaction from a vector such aspRL-CMV. The amplified renilla luciferase gene is ligated downstream of the POT1 promoter of pRS305 to obtain renilla luciferase construct (Vector II).
• the Potl promoter is PCR amplified from P. pastoris genomic DNA.
• the amplicon is subjected to agarose gel electrophoresis against a suitable marker.
• the amplified 500bp on the gel is eluted using Qiagen spin columns.
• the digested vector is ligated with amplified POT1 promoter. Further, the firefly luciferase gene with the peroxisomal targeting signal (SKL) is amplified by polymerase chain reaction from the vector such as plasmid pRSVL. The amplified firefly luciferase gene is ligated downstream of the POT1 promoter of pIBl to obtain firefly luciferase construct (Vector III).
• the Potl promoter is PCR amplified from P. pastoris genomic DNA.
• the amplicon is subjected to agarose gel electrophoresis against a suitable marker.
• the amplified 600bp on the gel is eluted using Qiagen spin columns.
• the digested vector is ligated with amplified POTl promoter.
• the renilla luciferase gene is amplified by polymerase chain reaction from a vector such as plasmid pRL-CMV, the amplified Renilla luciferase gene is ligated downstream of the POTl promoter of pJCF-214 to obtain renilla luciferase construct (Vector IV).
• sequences of the vectors- pPMlO and pPM5 comprising firefiy luciferase and Renilla luciferase genes respectively, which are expressed in Saccharomyces cerevisiae are as follows: pPM10(6240 bp)
• Renilla luciferase ORF 3434-4369 (in bold)
• Renilla luciferase ORF (in bold)
• Example 2 Strain constructions Strain construction in Saccharomyces cerevisiae
• Vector I and II are transformed into S. cerevisiae haploid strains including wild-type (WT), and the atgl (systematic gene name, YGL180W), atg5 (YPL149W) and atg8 (YBL078C) deletion strains (Gietz and Woods 2002).
• WT wild-type
• atgl systematic gene name, YGL180W
• YPL149W atg5
• YBL078C atg8 deletion strains
• the wild type and the deletion mutants are from the MATa collection, created by the Saccharomyces Genome Deletion Project. These strains are blocked in all autophagy related-pathways, including pexophagy.
• Transformation is carried out sequentially using the standard Lithium acetate transformation or electroporation procedure as below and the correct clones are selected for uracil (pFirefly- plasmid containing firefly luciferase gene) and leucine (pRenilla- plasmid containing renilla luciferase gene) auxotrophy, respectively.
• Vector III and IV are transformed into P.pastoris haploid strains including wild-type (WT, PPY12). Transformation is carried out using the electroporation procedure as mentioned below and the correct clones are selected for histidine (pFirefly) and arginine (pRenilla) auxotrophy, respectively.
• WT, atglA, atg5A, atgSA cells are grown to mid exponential phase in yeast nitrogen base (YNB without amino acids without ammonium sulphate- HIMEDIA, Catalogue number M151-100G)) with Ura and Leu dropout medium and transferred to oleate medium (0.1% oleate, 0.5%> Tween-40, 0.25%> yeast extract, 0.5%> peptone and 5mM phosphate buffer)for the induction of peroxisomes.
• yeast nitrogen base YNB without amino acids without ammonium sulphate- HIMEDIA, Catalogue number M151-100G
• Ura and Leu dropout medium and transferred to oleate medium (0.1% oleate, 0.5%> Tween-40, 0.25%> yeast extract, 0.5%> peptone and 5mM phosphate buffer
• the cell suspension is subjected to lysis using passive lysis buffer (PLB) at a pH of 7 and dual luciferase assay is performed on the cell lysate with the dual luciferase reporter assay system (Promega Corp., Madison, WI) according to the manufacturer's instructions at different time interval to analyse the firefly luciferase stability in PLB. Further, 10 ⁇ of luciferin i.e.
• the firefly luciferase substrate (from Promega Dual luciferase reporter assay system- Catalogue number El 980) is added immediately to the cell lysate and the luciferase count is taken after every 30 second to analyse the decay of firefly luciferase with respect to time ( Figure 6).
• the data is used to calculate the ratio of peroxisomal firefly luciferase to cytosolic Renilla luciferase activity.
• the rest of the lysate is also analysed by immunoblotting with anti-Potl antibodies.
• Firefly luciferase activity decreases drastically within a few minutes after addition of substrate. The rate of decline in relative firefly luciferase activity is compared to that of Potl levels in course of starvation induction. Based on preliminary data and understanding of pexophagy, it is understood that the firefly luciferase activity will reflect pexophagy as indicated by Potl levels. When compared to firefly luciferase activity it was observed that the decrease in Renilla luciferase activity was much slower than the firefly luciferase activity.
• Example 4 Pexophagy Assay
• POTl peroxisomal thiolase
• a peroxisomal matrix marker is used to assay pexophagy.
• the vacuolar processing of POT 1 -GFP occurs to yield stable GFP in the vacuole.
• This assay used two standards to measure pexophagy: 1) Level of thiolase degradation and 2) Level of GFP accumulation.
• Wild type POT 1 -GFP strain is a laboratory strain with genomically tagged GFP to the C terminus of Potl (HIS selection marker) obtained from Dr. Rachubinsky. Wild type BY4741 and all knockout strains are obtained from European Saccharomyces Cerevisiae Archive for Functional Analysis (EUROSCARF).
• the POT1-GFP-HIS cassette is amplified by using the following sets of primers:
• Wild type and atgl cells were transformed with POT1-GFP-HIS cassette by lithium acetate method. Transformants are screened by western blotting.
• Potl-GFP positive strains are allowed to grow till the A 60 o reaches 0.8-1 in YPD.
• Peroxisome biogenesis is induced by growing these cells in oleate medium for 12 hours. Cells are harvested, washed twice to remove traces of oleate and transferred to starvation medium without nitrogen, at inoculum density A 6 oo 3, to induce pexophagy. Cells are collected at various time intervals after pexophagy induction and processed by TCA method as below.
• Total cell lysates are electrophoresed on 12%>, SDS-PAGE for POT1-GFP processing pexophagy and transferred onto PVDF membrane at constant current of 2 Ampere for 30 minutes (Transblot turbo, BIORAD Inc, USA). Transfer is confirmed by Ponceau S staining of blot and the blots scanned and are used as loading controls. Blots are incubated overnight with 5% skim milk in primary anti-GFP mouse IgG antibody (Roche Diagnostics) at 1 : 3000 dilution). Secondary antibody used at 1 : 10,000 is goat anti-mouse conjugated to HRP (Biorad). Blots are developed by using ECL substrate and images captured using auto capture program in Syngene G-Box, UK. Image J (NIH) is used for quantitation of band intensities.
• Example 6 Identification of small molecule modulators of autophagy
• the dual luciferase assay is appropriate to identify small molecule modulators of the autophagy process. It is a kinetic assay which detects the decrease in the luciferase activity over time through autophagy. Therefore, through this assay the modulators of the autophagy pathway are detected in a high throughput format.
• a set of 13 cyclic peptoids represented as compounds 4a to 4m are investigated for their ability to affect the autophagy process in Pichia Pastoris.
• one cyclic peptoid represented as compound (4a) is detected that enhances the rate of autophagy which is further validated by microscopy.
• the said compound also shows a dose dependent increase in the rate of degradation of the luciferase reporter over time (Figure 5).
• the luciferase based autophagy assay is done in the yeast P. pastoris, where degradation of cargo i.e. firefly luciferase is followed over time (upto 2 hours) upon induction of autophagy.
• Compounds are pre-added to the wells of 96 well plate. After transferring the transformed cells to starvation condition (SD-N media), the cells are then added to the plate already containing the compounds. Time taken for 50% decrease in cargo activity is plotted for untreated cells and the compounds at 50 ⁇ concentration for cyclic peptoids. Triplicate values for the control are plotted and a difference of 3 standard deviation units between the test and control is considered as significant.
• the luciferase assay can be done in a plate format and for shorter time durations, it is highly amenable for high throughput studies for the detection of small molecule modulators of autophagy.
• the dual luciferase assay can be employed to identify slow and fast degraders of peroxisomes; mutants which are partially blocked and completely blocked in pexophagy by comparing the rates of degradation of luciferase reporter of the mutant with the wild type cells.

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