Abstract

   [34]graphic file with name ao0c03420_0009.jpg

   Parkinson’s disease (PD) is a progressive neurodegenerative disorder,
   whose treatment with modern therapeutics leads to a plethora of side
   effects with prolonged usage. Therefore, the management of PD with
   complementary and alternative medicine is often pursued. In the
   Ayurveda system of alternative medicine, Yashtimadhu choorna, a Medhya
   Rasayana (nootropic), prepared from the dried roots of Glycyrrhiza
   glabra L. (licorice), is prescribed for the management of PD with a
   favorable outcome. We pursued to understand the neuroprotective effects
   of Yashtimadhu choorna against a rotenone-induced cellular model of PD
   using differentiated IMR-32 cells. Cotreatment with Yashtimadhu choorna
   extract rescued rotenone-induced apoptosis and hyperphosphorylation of
   ERK-1/2. Quantitative proteomic analysis of six peptide fractions from
   independent biological replicates acquired 1,561,169 mass spectra,
   which when searched resulted in 565,008 peptide-spectrum matches
   mapping to 30,554 unique peptides that belonged to 4864 human proteins.
   Proteins commonly identified in biological replicates and >4 PSMs were
   considered for further analysis, leading to a refined set of 3720
   proteins. Rotenone treatment differentially altered 144 proteins (fold
   ≥1.25 or ≤0.8), involved in mitochondrial, endoplasmic reticulum, and
   autophagy functions. Cotreatment with Yashtimadhu choorna extract
   rescued 84 proteins from the effect of rotenone and an additional
   regulation of 4 proteins. Network analysis highlighted the interaction
   of proteins and pathways regulated by them, which can be targeted for
   neuroprotection. Validation of proteomics data highlighted that
   Yashtimadhu confers neuroprotection by preventing mitochondrial
   oxidative stress and apoptosis. This discovery will pave the way for
   understanding the molecular action of Ayurveda drugs and developing
   novel therapeutics for PD.

Introduction

   Neurodegeneration is a progressive phenomenon at old ages, which occurs
   as a result of neuronal loss or the inability of neurons to transmit
   the signals. Parkinson’s disease (PD) is one such age-related
   progressive neurodegenerative disorder whose prevalence accounts for 3%
   of the population, above 65 years.^[35]1 PD is caused by loss of
   dopaminergic neurons from the substantia nigra pars compacta (SNpc),
   resulting in loss of the neurotransmitter dopamine, and leads to the
   development of motor symptoms. The death of dopaminergic neurons is
   caused as a result of mitochondrial dysfunction, endoplasmic reticulum
   (ER) stress, neuroinflammation, and accumulation of protein
   aggregates.^[36]2,[37]3 The management of PD relies on the alleviation
   of the symptoms, and levodopa is one of the most commonly prescribed
   medicines, which is useful in the early stages of the disease. However,
   with prolonged usage, the efficacy of PD medications decline and is
   reported to cause many side effects.^[38]4 Thus, there is a need for
   the use of a sustainable alternative and complementary management
   strategy of PD, which is required not only to prevent symptoms of PD
   but also to address the underlying molecular aberrations and improve
   the quality of life.

   The quest for sustainable management of the disease led to the
   exploration of complementary and alternative medicines, which employ
   the use of traditional medicines and other forms of alternative
   treatments, including acupuncture, rhythmic therapy, and Reiki.^[39]5
   The Indian Ayurvedic medicinal system classifies medicinal plants with
   nootropic properties as Medhya Rasayana, which helps in improving
   memory and brain functions.^[40]6−[41]8 The nootropic formulations
   include Yashtimadhu (Glycyrrhiza glabra, licorice), Mandukaparni
   (Centella asiatica, Asiatic pennywort), Ashwagandha (Withania
   somnifera, Indian ginseng), and Brahmi (Bacopa monnieri, water
   hyssop).^[42]9−[43]11 Yashtimadhu choorna is prepared from the dried
   roots of G. glabra L., commonly known as licorice
   ([44]http://www.theplantlist.org/;
   [45]http://www.ayurveda.hu/api/API-Vol-1.pdf). Several studies have
   reported its effectiveness as a neuroprotectant, an antidepressant, an
   antioxidant, and a memory enhancer.^[46]12−[47]18 These studies
   highlight the neuromodulatory effects of Yashtimadhu; however, there
   exist lacunae in understanding the regulation of protein networks and
   the underlying molecular pathways.

   PD is regarded as proteinopathy^[48]19,[49]20 because of the
   dysregulation of the protein dynamics, resulting in abnormal
   accumulation of protein aggregates. Mass spectrometry (MS)-based
   proteomics serves as an invaluable tool to study the alteration of
   proteins that drive the pathogenesis in experimental models of PD, and
   the counteracting neuroprotective strategies, upon the introduction of
   a specific drug. The experimental models of PD are generated using
   genetically modified in vivo models such as α-synuclein (SNCA), parkin
   (PRKN), leucine-rich repeat kinases (LRRKs), or use of neurotoxins in
   vitro and in vivo.^[50]21 The commonly used neurotoxic models of PD are
   generated using rotenone, MPTP
   (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine), paraquat, and
   6-hydroxydopamine.^[51]22 The establishment of an in vitro cellular
   model of PD offers advantages in monitoring and understanding the
   cellular mechanisms that are responsible for neurodegeneration. These
   proteins involved in the neurodegenerative mechanism can be targeted
   for efficient neuroprotective interventions.^[52]23,[53]24 The study of
   proteins altered during the progression of a disease and its recovery
   helps in discovering the proteins associated with a protective pathway
   conferred by the drug.

   In this study, we sought to evaluate the neuroprotective effects of
   Yashtimadhu choorna extract in an in vitro PD model. This model was
   generated using rotenone (a mitochondrial complex-I inhibitor), which
   triggers oxidative stress and induces apoptosis,^[54]25 effectively
   mimicking the cellular aberrations reported in PD. Quantitative
   proteomics was employed to identify proteins altered by rotenone and
   compared to their restoration by Yashtimadhu choorna extract to
   understand the molecular networks involved in neuroprotection.

Results and Discussion

Yashtimadhu Choorna Extract Counteracts Rotenone-Induced Apoptosis

   We aimed to evaluate the establishment of the rotenone-induced PD model
   and the neuroprotection conferred by Yashtimadhu. To achieve this, we
   used IMR-32 cells that were differentiated with retinoic acid to yield
   dopaminergic neuron population.^[55]26−[56]28

   IMR-32 cells were differentiated with retinoic acid, which was
   confirmed with tyrosine hydroxylase (TH) expression ([57]Supporting
   Information Figure S1A,B) and used to assess the neuroprotection
   conferred by Yashtimadhu choorna extract in a rotenone-induced PD
   model. Cytotoxicity assay was used to test the effective treatment
   doses of rotenone and Yashtimadhu choorna extract. Rotenone-induced
   time- and dose-dependent cell death and the IC[50] (50% cell death)
   value was observed at a 100 nM concentration for 48 h, which was used
   for further treatments ([58]Supporting Information Figure S1C). At the
   same time, treatment with Yashtimadhu choorna extract for 48 h showed
   no cytotoxicity at all tested concentrations ([59]Supporting
   Information Figure S1D). The cell death observed at the IC[50] rotenone
   concentration of 100 nM was prevented by cotreatment of Yashtimadhu
   choorna extract at a 200 μg/mL concentration, which was used for
   further treatments ([60]Figure [61]1
   A). Cell death was also ascertained with the live–dead cell staining
   assay ([62]Figure [63]1B,C). Rotenone treatment increased cell death
   (7.53-fold, p < 0.001), which was rescued by Yashtimadhu choorna
   extract cotreatment (0.89-fold, p < 0.001), which is in agreement with
   the cytotoxicity analysis.

Figure 1.

   [64]Figure 1
   [65]Open in a new tab

   PD model validation and Yashtimadhu neuroprotection. (A) Cytotoxicity
   analysis showing rotenone-induced cell death and prevention of cell
   death by Yashtimadhu choorna extract cotreatment; (B) live–dead cell
   staining assay using propidium iodide (PI) (red) and HOECHST (blue)
   nuclear counterstain; and (C) graphical representation of cell death
   with respect to untreated cell control. Rotenone treatment increases
   cell death, encountered by Yashtimadhu choorna cotreatment, (D)
   immunoblot analysis of p-ERK-1/2 (T202/T204) and cleaved caspase-3 with
   β-actin as the loading control across control (C), rotenone (R),
   rotenone + Yashtimadhu choorna cotreatment (RY), and Yashtimadhu
   choorna extract (Y); (E) densitometry graph of cleaved caspase-3
   activation, normalized with loading control; and (F) densitometry graph
   of p-ERK-1/2 activation, normalized with loading control, *, with
   respect to control (C) and # with respect to Yashtimadhu choorna
   cotreatment (RY). *,# p < 0.05, **,## p < 0.01, and ***,### p < 0.001.
   Labels: C-untreated cells, R-rotenone treatment, RY—rotenone +
   Yashtimadhu choorna extract cotreatment, and Y—Yashtimadhu choorna
   extract treatment.

   ERK-1/2 phosphorylation and cleaved-caspase-3 activation are known to
   be induced by rotenone, which is also implicated in
   neurodegeneration.^[66]29,[67]30 Rotenone induced activation of cleaved
   caspase-3 (2.22-fold, p < 0.05) and hyperphosphorylation of ERK-1/2
   (1.87-fold, p < 0.05) ([68]Figure [69]1
   D–F). Yashtimadhu choorna extract cotreatment encountered the
   rotenone-induced caspase-3 activation (0.88-fold, p < 0.05) and
   p-ERK-1/2 (0.58-fold, p < 0.01). These observations confirm the
   neuroprotective effects of Yashtimadhu against rotenone-induced stress
   by preventing p-ERK-1/2 activation and subsequent apoptosis.

Quantitative Proteomics Highlights Differentially Regulated Proteins

   Quantitative proteomics was carried out to identify the altered
   proteins with rotenone treatment alone and restoration of those by
   cotreatment with Yashtimadhu choorna extract. The data were acquired
   for the biological duplicates, as technical triplicates each
   ([70]Figure [71]2
   A). The workflow used for filtering proteins based on their
   identification from the independent replicates is outlined in
   [72]Figure [73]2B. We obtained around 1.5 million tandem mass
   spectrometry (MS/MS) spectra, which provided 565,008 PSMs upon
   searching the MS/MS data against the database (human RefSeq109). These
   PSMs corresponded to 30,554 nonredundant peptides, identifying 4864
   protein sequences corresponding to 4783 genes. Proteins commonly
   identified in both the biological replicates were considered, resulting
   in 3762 proteins. Also, proteins with PSMs < 4 were filtered out, and a
   list of 3720 resultant proteins (corresponding to 3673 gene symbols)
   was used for subsequent analysis ([74]Supporting Information Table S1).
   The fold change (FC) of these 3720 proteins, along with their
   respective p-values and q-values, is deposited in the PRIDE repository.

Figure 2.

   Figure 2
   [75]Open in a new tab

   Summary of proteomics analysis. (A) Schematic of workflow employed for
   proteomics data acquisition. Labels: C-untreated cells, R-rotenone
   treatment, RY—rotenone + Yashtimadhu choorna extract cotreatment, and
   Y—Yashtimadhu choorna extract treatment and (B) schematic of the
   workflow used for proteomics data analysis.

   Principal component analysis (PCA) was carried out to identify the
   variance across the four groups. The score plot ([76]Figure [77]3
   A) shows the clustering of the groups based on the biological
   replicates. Rotenone treatment showed the maximal variation in
   comparison with the untreated cell control group. In contrast, the
   Yashtimadhu choorna extract cotreatment group showed a minimum variance
   compared with the untreated cells, indicating the restoration of
   altered protein dynamics.

Figure 3.

   [78]Figure 3
   [79]Open in a new tab

   Differential regulation of proteins. (A) PCA score plot showing the
   variability between the groups and within the biological replicates,
   (B) scatter plot showing rotenone-induced differential expression, and
   (C) Venn diagram showing the restoration of proteins with Yashtimadhu
   choorna cotreatment. Labels: C-untreated cells, R-rotenone treatment,
   RY—rotenone + Yashtimadhu choorna extract cotreatment, and
   Y—Yashtimadhu choorna extract treatment.

   The FC of protein expression was calculated with respect to the
   untreated cell control (C) and rotenone (R). A FC cutoff of ≥1.25 for
   overexpression and ≤0.8 for the downregulation of proteins was selected
   to be biologically significant, and an adjusted p-value, that is, q ≤
   0.05, was selected for statistical significance. A similar range of FCs
   has been previously reported by studies using isobaric
   labeling.^[80]31−[81]35 Rotenone treatment highlighted 421
   differentially regulated proteins (FC ≥1.25 or ≤0.8), of which 144 were
   significantly altered (q ≤ 0.05, with respect to untreated cell
   control, [82]Figure [83]3
   B). Yashtimadhu choorna extract cotreatment was found to significantly
   regulate 88 proteins (q ≤ 0.05, with respect to rotenone), which
   included restoration of 84 proteins that were dysregulated by rotenone
   and an additional regulation of 4 proteins ([84]Figure [85]3C).

Classification of Identified Proteins

   Proteins were classified based on their intracellular
   compartmentalization and biological processes to understand the
   relevance of the differentially regulated proteins in the context of
   PD. Proteins were localized in the cytoplasm and nucleus, followed by
   exosomes, mitochondria, lysosomes, and ER. The proteins identified were
   also involved in processes such as cellular and protein metabolism,
   energy pathways, and cell proliferation.

   Additionally, proteins were also classified using publicly available
   databases, such as MitoCarta, The Autophagy database, and the
   E3-ligases databases. The proteins regulating mitochondrial processes
   and energy pathways, ER function, autophagy, metabolism of proteins and
   lipids, cell proliferation, DNA repair, and replication were further
   shortlisted to understand the effect of rotenone and rescue by
   Yashtimadhu choorna extract cotreatment.

Rotenone Dysregulates Proteins Involved in Vital Functions

   The extent of cellular damage as a result of rotenone was earlier
   ascertained with molecular assays, and the proteomics data were used to
   gain deeper insights into rotenone-induced cellular stress. Rotenone
   treatment resulted in 30 overexpressed (≥1.25, q < 0.05) and 114
   downregulated (≤0.8, q < 0.05) proteins ([86]Figure [87]4
   A). Mitochondrial dysfunction, ER stress, and autophagy dysregulation
   drive neuronal cell death, leading to the onset and progression of
   PD.^[88]36−[89]38 Rotenone treatment altered the proteins involved in
   mitochondrial functions, ER functions, and autophagy. Altered
   mitochondrial proteins include those involved in the tricarboxylic acid
   (TCA) cycle and oxidative phosphorylation (OxPhos), among others
   ([90]Figure [91]4B). The expression of these classified proteins with
   respect to control is also depicted in the chord diagram ([92]Figure
   [93]4B). A heat map showing the expression of the highly overexpressed
   (FC ≥1.5, q ≤ 0.05) and downregulated ( FC ≤0.67, q ≤ 0.05) proteins
   with rotenone treatment is given in [94]Figure [95]4C.

Figure 4.

   [96]Figure 4
   [97]Open in a new tab

   Rotenone-induced differential protein expression. (A) Volcano plot
   displaying the differentially altered proteins with rotenone treatment,
   with respect to control group (R vs C), (B) chord plot showing the
   classification of differentially regulated proteins and their log 2FC,
   (C) heat map showing the differential expression of the highly
   upregulated (FC ≥ 1.5) and downregulated (FC ≤ 0.67) proteins, with
   respect to that of the control group. Labels: C-untreated cells and
   R-rotenone treatment.

   OxPhos proteins such as ubiquinol-cytochrome c reductase hinge protein
   (UQCRH), NADH: ubiquinone oxidoreductase subunit-AB1 (NDUFAB1), and TCA
   cycle protein, dihydrolipoamide dehydrogenase were downregulated.
   Rotenone also affected the expression of ER proteins such as signal
   recognition particle 9 (SRP9), calumenin (CALU), eukaryotic translation
   initiation factor 2B subunit delta (EIF2B4), and autophagy proteins,
   1-phosphatidylinositol 4,5-bisphosphate phosphodiesterase eta-1 (PLCH1)
   and cathepsin D (CTSD). The findings indicate mitochondrial
   dysfunction, autophagy dysregulation, and ER stress, which, as a result
   of rotenone, is reported to be a driving factor in neuronal
   apoptosis.^[98]25,[99]39,[100]40

Yashtimadhu Restores Proteins Involved in Cellular Stress Response

   We have compared the Yashtimadhu choorna extract cotreatment group with
   the rotenone treatment group for differentially regulated proteins,
   which have been highlighted in a volcano plot ([101]Figure [102]5
   A). The differentially regulated proteins are involved in mitochondrial
   functions, including the proteins in OxPhos and TCA cycle, ER and
   autophagy functions, cell proliferation, DNA repair, and DNA
   replication ([103]Figure [104]5B). The proteins differentially
   regulated with Yashtimadhu choorna extract cotreatment are listed in
   [105]Table [106]1 and heat map ([107]Figure [108]5C).

Figure 5.

   [109]Figure 5
   [110]Open in a new tab

   Yashtimadhu choorna extract cotreatment restores differentially
   regulated proteins. (A) Volcano plot displaying the differentially
   altered proteins with Yashtimadhu choorna extract cotreatment with
   rotenone, with respect to the rotenone group (RY vs R), (B) Sankey
   diagram showing the classification of differentially regulated
   proteins, and (C) heat map showing the differential expression of the
   proteins classified across all four groups, with respect to control.
   Labels: C-untreated cells, R-rotenone treatment, RY—rotenone +
   Yashtimadhu choorna extract cotreatment, and Y—Yashtimadhu choorna
   extract treatment.

Table 1. Partial List of Proteins Regulated by Yashtimadhu with Their
Respective FC and q-Values[111]^a.

   description accession gene symbol rotenone, FC Yashtimadhu choorna
   extract cotreatment, FC
   Proteins Restored by Yashtimadhu
   Proteins Overexpressed with Rotenone
   pygopus homolog 2 [112]NP_612157.1 PYGO2 5.09,* 0.96,#
   X-ray radiation resistance-associated protein 1 isoform X10
   [113]XP_011543064.1 XRRA1 2.08,* 1.4,#
   bromodomain-containing protein 9 isoform 1 [114]NP_076413.3 BRD9
   1.94,** 1.43,#
   10 kDa heat shock protein, mitochondrial [115]NP_002148.1 HSPE1 1.34,*
   1.03,#
   UDP-N-acetylhexosamine pyrophosphorylase-like protein 1 isoform X1
   [116]XP_006717380.1 UAP1L1 1.39,* 0.99,#
   1-phosphatidylinositol 4,5-bisphosphate phosphodiesterase eta-1 isoform
   X5 [117]XP_011510867.1 PLCH1 1.41,* 0.93,#
   anillin isoform X1 [118]XP_006715809.1 ANLN 1.41,* 1.13,#
   ornithine aminotransferase, mitochondrial isoform 1 [119]NP_001309897.1
   OAT 1.38,* 1.31,#
   tripartite motif-containing protein 65 isoform X1 [120]XP_006721823.1
   TRIM65 1.35,* 0.89,#
   kallikrein-11 isoform X1 [121]XP_011524671.1 KLK11 1.32,* 0.76,#
   Proteins Downregulated with Rotenone
   60S ribosomal protein L37 [122]NP_000988.1 RPL37 0.67,* 0.89,#
   superoxide dismutase 1 [Cu–Zn] [123]NP_000445.1 SOD1 0.74,* 0.77,#
   zinc finger Ran binding domain-containing protein 2 isoform 1
   [124]NP_976225.1 ZRANB2 0.76,* 0.85,#
   histone H1.3 [125]NP_005311.1 H1-3 0.76,*** 0.84,#
   CD59 glycoprotein [126]NP_001120699.1 CD59 0.76,*** 0.89,#
   nucleolin [127]NP_005372.2 NCL 0.77,* 0.83,#
   CALU isoform c [128]NP_001186600.1 CALU 0.77,** 0.88,#
   Src substrate cortactin isoform a [129]NP_005222.2 CTTN 0.78,***
   0.84,###
   SH3 domain binding glutamic acid-rich-like protein [130]NP_003013.1
   SH3BGRL 0.79,** 0.86,#
   ashwin isoform X1 [131]XP_024308903.1 C2orf49 0.79,** 0.89,#
   Proteins Additionally Regulated by Yashtimadhu
   leucine-rich repeat-containing protein 14 [132]NP_001258965.1 LRRC14
   1.51,ns 0.97,#
   pleckstrin homology-like domain family A member 3 [133]NP_036528.1
   PHLDA3 1.02,ns 0.72,#
   neurabin-2 [134]NP_115984.3 PPP1R9B 0.95,ns 1.24,#
   CD63 antigen isoform A [135]NP_001244318.1 CD63 0.86,ns 1.16,#
   [136]Open in a new tab
   ^a

   *, significance with respect to control; #, significance with respect
   to rotenone. *,#, q ≤ 0.05; **,##, q ≤ 0.01; and ***,###, q ≤ 0.005.

Yashtimadhu Regulates Pathways Essential for Neuronal Maintenance

   Protein dynamics play a crucial role in the regulation of several
   cellular pathways. From the pathway enrichment analysis using the
   Enrichr and Reactome tools, significant pathways were identified (p <
   0.05) and were narrowed down to essential pathways, such as essential
   neuronal functions, metabolism of proteins, and metabolism of lipids.
   The proteins involved in these pathways are summarized in [137]Table
   [138]2
   . Regulation of apoptosis, dopaminergic synapse, superoxide removal,
   mitochondrial functions, maintenance of myelin, axonal transport, and
   postsynaptic differentiation was enriched. In addition to this, the
   regulation of lipid and protein metabolism was also enriched.

Table 2. Cellular Pathways Regulated by Yashtimadhu.

   pathway name proteins
   Neuronal Processes
   dopaminergic synapse CALM3
   axon guidance SDC2; CLTB; CLTA; RPL37
   transmission across chemical synapses PRKAR1A; CALM3
   neurotransmitter receptors and postsynaptic signal transmission
   PRKAR1A; CALM3
   long-term potentiation CALM3
   agrin in postsynaptic differentiation CTTN
   synaptic vesicle cycle CLTB; CLTA
   regulation of mitotic cell cycle SKP1
   regulation of cell cycle NASP SKP1
   mitochondrial transcription SSB
   ROS degradation SOD1
   apoptotic DNA fragmentation HMGB2; HMGB1
   Protein Metabolism
   protein metabolism EIF4B; HSPE1; SRSF1; NACA; TBCA; SERPINA3; SKP1;
   RPL37; KLK11
   translation initiation complex formation EIF4B
   cap-dependent translation initiation RPL37; EIF4B
   eukaryotic translation elongation and termination RPL37
   protein processing in the ER SKP1
   protein folding TBCA
   UPR HDGF
   activation of chaperones by IRE1α HDGF
   autophagy HMGB1; PLCH1, CTLA, CTLB
   neddylation DCUN1D3
   Lipid Metabolism
   metabolism of lipids PSAP; CYP51A1; AGPAT1
   sphingolipid and glycosphingolipid metabolism PSAP
   phospholipid and glycerophospholipid metabolism AGPAT1
   CDP-diacylglycerol biosynthesis AGPAT1
   cholesterol biosynthesis CYP51A1
   [139]Open in a new tab

Yashtimadhu Prevents Mitochondrial Dysfunction

   Mitochondrial dysfunction is attributed to increased oxidative stress
   and loss of mitochondrial membrane potential and results in activation
   of the apoptotic cascade in neurons.^[140]41 The mitochondrial OxPhos
   complex-III protein, cytochrome b-c1 complex subunit 6 (UQCRH), plays a
   vital role in ATP synthesis and maintenance of mitochondrial membrane
   potential. Gene expression analysis points to the reduction of UQCRH in
   the SNpc dopaminergic neurons of PD patients,^[141]42 correlating with
   the observation from rotenone treatment, which was increased with
   Yashtimadhu choorna cotreatment. Yashtimadhu also restored ATP synthase
   inhibitory factor subunit 1 (ATP5IF1) and superoxide dismutase-1
   (SOD1), which are reported to be downregulated with rotenone.^[142]43
   SOD1 quenches the reactive oxygen species (ROS) and maintaining redox
   balance, and ATP5IF1 inhibits mitochondrial ATP synthase, that is,
   complex-V and maintains the mitochondrial membrane
   potential.^[143]44,[144]45 The reduction in ATP5IF1 is implicated in
   mitochondriopathies, while its overexpression confers
   neuroprotection.^[145]45,[146]46 Yashtimadhu also countered
   rotenone-induced increase of chaperonin, that is, heat shock protein-10
   kDa (HSPE1) that helps in the folding of mitochondrial
   proteins.^[147]47 Rotenone-mediated increase of HSPE1 is reported to be
   involved in caspase-3 activation and neuron death.^[148]48,[149]49 From
   these observations, it can be inferred that Yashtimadhu prevented
   cleaved caspase-3-mediated apoptosis by restoring the mitochondrial
   proteins involved in redox balance, membrane potential, and regulation
   of apoptosis.

Yashtimadhu Restores Proteins Involved in Lipid Metabolism

   The differentially expressed proteins with Yashtimadhu choorna extract
   cotreatment were found to be involved in the metabolism of
   phospholipid, sphingolipid, cytidine diphosphate diacylglycerol
   (CDP-DAG), and cholesterol metabolism. The proteins enriched were
   cytochrome 1-acyl-sn-glycerol-3-phosphate acyltransferase 1 (AGPAT1),
   P450 family 51 subfamily A member 1, that is, lanosterol 14-alpha
   demethylase (CYP51A1), and prosaposin (PSAP). AGPAT1 is a
   lysophosphatidic acid acyltransferase that regulates phospholipid and
   triglyceride levels in the brain, while CYP51A1 is involved in
   cholesterol metabolism.^[150]50,[151]51 Dysregulated lipid metabolism
   is associated with the cytoplasmic accumulations of SNCA with other
   proteins and lipids, leading to the formation of protein aggregates,
   that is, Lewy bodies in PD.^[152]52 Lipidomic analysis of PD brains has
   demonstrated an increase in cholesterol metabolism and dysregulation of
   glycerophospholipid (CDP-DAG),^[153]53 signifying the classification of
   PD as a lipidopathy.^[154]19 The perturbations of lipid metabolism as a
   result of rotenone exposure in SHSY5Y cells have also been
   reported.^[155]54 The restoration of the proteins regulating lipid
   metabolism highlights the protective effect of Yashtimadhu.

Interplay between Protein Translation and Degradation Mechanisms by
Yashtimadhu

   In our analysis, we also enriched the proteins involved in protein
   translation and degradation. ER plays a central role in protein
   dynamics in the cell, regulating the translation, protein folding,
   trafficking, and unfolded protein response (UPR).^[156]55 Yashtimadhu
   restored ER proteins involved in translation and protein folding such
   as eukaryotic translation initiation factor (EIF4B) and translational
   regulator 60S ribosomal protein L37 (RPL37). Protein folding is a major
   determinant of its function, and with the accumulation of unfolded or
   misfolded proteins, oxidative stress and ER stress are induced. The ER
   stress results in the activation of UPR, which plays an essential role
   in PD.^[157]56,[158]57 In response to the accumulation of unfolded
   proteins, the UPR regulates signals that activate the protein
   degradation pathways, that is, autophagy and ubiquitin-proteasomal
   system (UPS).^[159]57,[160]58

   Autophagy is a bulk degradation mechanism that degrades proteins and
   damaged cell organelles. Autophagy initiation is controlled primarily
   by mammalian target of rapamycin (mTOR) and 5′ AMP-activated protein
   kinase (AMPK), which in turn regulates the phosphorylation of
   ULK1/2.^[161]59,[162]60 UPS is involved in clearing protein aggregates,
   which is carried out by the 26s proteasomal system and is reported to
   act in close association with the autophagy mechanism.^[163]61,[164]62
   Proteins that are tagged by the E3-ubiquitin ligases are selected for
   degradation.^[165]63 Yashtimadhu regulated autophagy proteins such as
   1-phosphatidylinositol-4,5-bisphosphate phosphodiesterase (PLCH1),
   clathrins (CTLA and CTLB), and high mobility group box 1 (HMGB1).
   E3-ligase-associated proteins, such as BTB/POZ domain-containing
   adapter for CUL3-mediated RhoA degradation (TNFAIP1), galectin-3
   binding protein (LGALS3BP), and tripartite motif-containing 65
   (TRIM65), were also restored by Yashtimadhu.

   The enrichment analysis identified selective protein degradation
   pathways such as autophagy and neddylation. Neddylation is a
   degradation process similar to ubiquitination that involves the
   addition of a NEDD8, whose activation has been proven to be protective
   in PD.^[166]64 Aggrephagy is a form of selective autophagy process that
   takes over the degradation of the accumulated proteins when the
   cellular UPS fails.^[167]65,[168]66 The dysregulation of the protein
   degradation mechanism leads to the accumulation of protein aggregates,
   which induce apoptosis as a result of oxidative and ER stress in PD.
   The regulation of the proteins involved in the translation and
   degradation processes makes Yashtimadhu an attractive candidate for
   neuroprotection.

Regulation of Transcription Factors by Rotenone and Yashtimadhu

   We enriched the transcription factors (TFs) that control the expression
   of the differentially altered proteins to understand the regulation of
   expression at the level of transcription ([169]Supporting Information
   Tables S2 and S3). The proteins altered by rotenone treatment were
   found to be regulated by 169 TFs. Similarly, the proteins restored by
   Yashtimadhu were found to be regulated by 149 TFs. A list of the top
   enriched TFs and the proteins regulated by them is displayed in
   [170]Table [171]3
   , which includes specificity proteins-1 and 4 (SP1 and SP4), early
   growth response-1 (EGR1), nuclear respiratory factor-1 (NRF1), and
   Kruppel-like factor 7 (KLF7), to name a few. Some of the enriched TFs
   were previously reported to be dysregulated in PD pathogenesis, such as
   EGR1,^[172]67,[173]68 NRF1,^[174]69 and ELK1.^[175]70 These TFs were
   found to regulate proteins involved in autophagy, energy pathways, and
   metabolism of proteins and lipids. The findings show that Yashtimadhu
   helps in the regulation of TFs, which in turn restores the proteins
   impacted by rotenone.

Table 3. List of TFs Regulating the Rotenone- and Yashtimadhu-Induced
Differentially Expressed Proteins.

   TF no. of proteins, regulated by rotenone no. of proteins, regulated by
   Yashtimadhu representative proteins
   SP1 64 39 NCL; CTTN; HMGB1; TPM3; PSAP; NASP; EIF4B; SOD1; HSPE1
   KLF7 45 31 FUS; PTMS; PDCD5; CD59; CIRBP; GAA; EWSR1; UQCRH
   SP4 39 21 HMGN2; HMGB2; SET; NUCKS1; CIRBP; ITGA3; CLTB
   EGR1 37 20 NASP; EIF4B; SRSF1; FUS; MARCKSL1; OAT; NACA
   NFYA 33 17 LRRC14; TAF15; PRKAR1A; C12orf57; CLTA; ZRANB2; XRRA1
   GABPA 27 16 TGOLN2; C12orf57; RPL37; CHCHD2; BICD2; C2orf49; BRD9
   NRF1 22 15 PSAP; SSB; SRSF1; MARCKSL1; TBCA; PAGE5; PDCD5; GAA
   YY1 22 15 CAPRIN1; TMSB4X; PRKAR1A; SRSF3; UQCRH; MORF4L1
   ELK1 22 12 MT2A; TGOLN2; CLTA; RRP15; RPL37; CHCHD2; TNFAIP1
   HNF4A 21 13 TPM3; HSPE1; FUS; HPCAL1; CALM3; SH3BGRL; CD59; ITGA3
   [176]Open in a new tab

Molecular Network Involved in the Neuroprotective Functions of Yashtimadhu

   The functional significance of the proteins regulated by Yashtimadhu
   cotreatment and their cellular functions were established, and the
   protein–protein interaction (PPI) network was constructed with STRING
   (confidence score ≥ 0.7). The PPI analysis showed the interactions
   between 31 out of the 84 proteins differentially regulated by
   Yashtimadhu ([177]Supporting Information Table S4). We manually curated
   the molecular pathways that are governed by the 31 proteins and rebuilt
   the interaction network, highlighting the PPI and their interconnected
   pathways ([178]Figure [179]6
   ).

Figure 6.

   [180]Figure 6
   [181]Open in a new tab

   Molecular network of Yashtimadhu-mediated neuroprotection. The
   interaction network of Yashtimadhu-regulated proteins and interlinking
   pathways.

   The pathways regulated by the PPI were maintenance of neuronal
   functions, protein translation, cellular signaling (receptor tyrosine
   kinases, G-protein signaling, PI3K-AKT, and mTOR signaling), ROS
   degradation, RNA metabolism, energy pathway, autophagy, and apoptosis
   regulation, while the proteins regulating lipid mechanism formed a
   unique cluster. The network provides a snapshot of the proteins and
   their functions, modulating the neuroprotective of Yashtimadhu, against
   the rotenone-induced in vitro model of PD.

Validation of Neuroprotection Conferred by Yashtimadhu

   The MS-based analysis highlighted the proteins and pathways regulated
   by Yashtimadhu, which were predominantly involved in mitochondrial
   functions. As stated earlier, mitochondria regulate several cellular
   functions such as energy production (TCA and OxPhos), maintenance of
   cellular redox, and apoptosis. We validated some of the proteins and
   pathways regulated by Yashtimadhu, which are involved in mitochondrial
   homeostasis.

   We selected enzymes that are involved in the regulation of cellular
   redox (SOD1), TCA cycle [pyruvate dehydrogenase E1 subunit α 1 (PDHA1),
   citrate synthase (CS), and isocitrate dehydrogenase 1 (IDH1)], and
   apoptosis [voltage-dependent anion-selective channel protein 1 (VDAC1)
   and apoptosis-inducing factor mitochondrial 1 (AIFM1)]. The abundance
   of these proteins from the MS-based proteomics is given in [182]Figure
   [183]7
   A. Immunoblotting analysis correlated with the proteomics analysis and
   showed a decrease in SOD1 with rotenone, which was subsequently
   restored by Yashtimadhu. PDHA1, AIFM1, and VDAC1 were also increased in
   rotenone that was restored by Yashtimadhu ([184]Figure [185]7B,C). CS
   and IDH1 did not show significant changes in their expression patterns
   across the treatment conditions. VDAC is located on the mitochondrial
   outer membrane, forming channels for the transport of metabolites. It
   also plays a prime role in apoptosis by facilitating the release of
   proapoptotic proteins such as cytochrome-C and AIFM1, initiating
   apoptosis.^[186]71 Rotenone-mediated increase in VDAC and AIFM1
   suggests activation of apoptosis as a result of mitochondrial
   dysfunction, which is prevented by Yashtimadhu.

Figure 7.

   [187]Figure 7
   [188]Open in a new tab

   Validation of proteins and pathways regulated by Yashtimadhu. (A) Heat
   map showing the FC abundances of selected proteins from MS data, (B)
   immunoblotting analysis of proteins involved in mitochondrial health,
   (C) densitometry analysis showing the expression of the proteins, (D)
   cellular ROS staining using DCFDA (green) and counterstain HOECHST
   (blue), and (E) graphical representation of ROS production in the
   cells. *, significant with respect to control; #, significant with
   respect to rotenone. *,#, p ≤ 0.05; **,##, p ≤ 0.01. Labels:
   C-untreated cells, R-rotenone treatment, RY—rotenone + Yashtimadhu
   choorna extract cotreatment, and Y—Yashtimadhu choorna extract
   treatment.

   Rotenone-induced reduction in SOD1 levels was reported earlier, which
   also led to increased oxidative stress.^[189]43 Our pathway analysis
   also pointed to the regulation of cellular ROS degradation. We used
   2′,7′-DCFDA (2′,7′-dichlorofluorescein diacetate) staining dye to
   observe the cellular ROS, which was increased with rotenone decreased
   with Yashtimadhu choorna extract cotreatment. Also, alteration in PDHA1
   is implicated in ER stress before induction of mitochondrial
   dysfunction,^[190]72 and an increase in PDHA1 is also associated with
   increased ROS production.^[191]73 Our observations of ROS correlated
   with the reduced SOD1 levels with rotenone, indicating that the action
   of Yashtimadhu is by modulating SOD1 and PDHA1 levels and regulating
   cellular ROS degradation. The Yashtimadhu-mediated reduction in
   cellular ROS prevents cell death by preventing mitochondrial
   dysfunction and subsequent release of apoptotic factors.

Conclusions

   This study provides invaluable information on the neuroprotective
   properties of Yashtimadhu choorna against rotenone-induced PD model
   using quantitative proteomics. Yashtimadhu restored functions that are
   dysregulated in PD, such as mitochondrial function, ER health, protein,
   and lipid metabolism. The findings of this study enabled us to
   understand the proteins involved in rotenone-induced neurotoxicity that
   was counteracted by Yashtimadhu. Analysis of the restored proteins
   enabled the identification of molecular networks involved in the
   neuroprotective functions of Yashtimadhu. Validation of the proteins
   and pathways regulated by Yashtimadhu shows that it confers
   neuroprotection by the restoration of cellular redox potential and
   maintaining mitochondrial health, thereby preventing subsequent
   neuronal apoptosis. The identified proteins can be developed as
   molecular targets for neuroprotection against PD.

   Our study opens new avenues in understanding and deciphering the
   molecular mechanisms of traditional medicine in combating
   neurodegenerative disorders. This omics-aided approach can be envisaged
   for molecularization of Indian Ayurvedic formulation-based therapeutic
   interventions.

Methods

Reagent Procurement

   Rotenone (Cat# R8875), retinoic acid (Cat# R2625), collagen (Cat#
   C9791), thiazolyl blue tetrazolium bromide (MTT, Cat# M5655),
   bisBenzimide H 33342 (HOECHST, Cat# B2261),
   2′,7′-dichlorodihydrofluorescein (DCFDA, Cat# D6883), iodoacetamide
   (Cat# I6125), and DL-dithiothreitol (Cat# D9779) were procured from
   Sigma-Aldrich, St. Louis, USA. Dulbecco’s modified Eagle medium (DMEM)
   high glucose (Cat#12100046), fetal bovine serum (FBS), and 100×
   antibiotic/antimycotic solution (Cat# 15240062) were purchased from
   Gibco. Pierce BCA protein estimation assay kit (Cat# 23225), Pierce
   Peptide estimation assay kit (Cat# 23275), and TMT 10plex kit (Cat#
   90110) were procured from Thermo Fisher Scientific USA. Antibodies were
   purchased from Cell Signaling Technology, Danvers, USA, and
   Sigma-Aldrich, St. Louis, USA. Nitrocellulose membrane (Cat# 1620115)
   and Clarity ECL Substrate (Cat# 170-5061) were purchased from BioRad
   Laboratories, California, USA, and X-ray films from Carestream, USA.
   TPCK-treated trypsin (Cat# [192]LS003741) was from Worthington
   Biochemical Corporation, USA. Solid-phase extraction disks, C-18 (Cat#
   66883-U), and SCX (Cat# 66889-U) were procured from Empore, USA.

Procurement of Yashtimadhu Choorna

   Yashtimadhu choorna (lot no.64) was procured from the SDP Remedies and
   Research Centre, Puttur, Karnataka, India
   ([193]http://sdpayurveda.com/products/choorna/yastimadhu-choorna/), a
   GMP-certified manufacturer of Ayurvedic formulations, and a specimen is
   maintained at the center with the identification number
   SDP/YM/001-2017. The industrial process includes shade-net drying and
   vacuum drum drying of Yashtimadhu roots, followed by pulverization of
   the dried roots and sieving, resulting in the fine powder, with a yield
   of 90%. Yashtimadhu choorna was procured, and the presence of lead
   molecules, glabridin (specific for G. glabra), glycyrrhizic acid, and
   licoricesaponin-G2 was confirmed using liquid chromatography–MS/MS
   (LC–MS/MS) analysis using QTRAP-6500, ABSCIEX. The compounds were
   identified based on the MS/MS fragment spectra, matched to the
   theoretical spectra generated using CFM-ID,^[194]74 and detailed in
   [195]Supporting Information Table S5.

Yashtimadhu Choorna Extract Preparation for Cell Culture Analysis

   Yashtimadhu choorna extract for cell culture treatment was prepared
   using an aqueous (water) extraction method. Yashtimadhu choorna, 1 g,
   was suspended in 10 mL of Milli-Q water (concentration, 0.1 g/mL) and
   incubated overnight at room temperature with continuous rotation. The
   extraction mixture was then centrifuged at 5000g for 10 min. The
   centrifugation was repeated for complete aspiration of the supernatant
   and transferred to a fresh tube. The aqueous extract was then dried
   using SpeedVac (Savant, Thermo Fisher Scientific, USA) and stored at
   −20 °C until further use. The dried extract was resuspended in
   serum-free cell culture media before treatment.

Cell Culture and Treatments

   IMR-32 cells (ATCC CCL-127) were procured from National Centre for Cell
   Science (NCCS), Pune, India. The cells were maintained in DMEM-high
   glucose supplemented with 10% FBS and 1× antibiotic/antimycotic
   solution and were incubated at 37 °C with 5% CO[2]. Differentiation of
   cells was carried out in a collagen-coated 6-well plate, wherein the
   cells were seeded at a density of 3 × 10^4 cells/well. The cells were
   treated with 10 μM retinoic acid supplemented in 2% FBS-containing
   medium, for 9 days, for yielding a dopaminergic population.
   Differentiation was confirmed with TH expression ([196]Supporting
   Information Figure S1A,B).

   For MTT assay, 5000 cells/well were seeded in a 96-well plate and
   treated with different concentrations of rotenone (0.25, 0.5, 1, 10,
   100, 200, 500, 1000, and 10000 nM) and Yashtimadhu choorna extract (50,
   100, 200, 500, 1000, and 1500 μg/mL) for 48 h. MTT dye was added to the
   cells and incubated for 4 h. The resultant formazan crystals were
   dissolved using 50:50 of ethanol/dimethyl sulfoxide (DMSO) solution and
   read at 570 nm and background subtraction at 650 nm. Cell viability was
   calculated and represented as a percentage with respect to the
   untreated cells. The concentrations of rotenone and Yashtimadhu were
   determined from the results of the cytotoxicity assay. The
   differentiated cells were treated with (i) 100 nM rotenone (dissolved
   in DMSO), (ii) 200 μg/mL Yashtimadhu choorna extract, and (iii) 100 nM
   rotenone + 200 μg/mL Yashtimadhu choorna extract for 48 h, and
   untreated cells were taken as a control.

Cell Staining Assays

   IMR-32 cells were seeded at 1 × 10^4 cells/well in 12-well plates,
   differentiated, and treated with rotenone (100 nM), Yashtimadhu choorna
   extract (200 μg/mL), 100 nM rotenone + Yashtimadhu choorna extract
   cotreatment for 48 h. The untreated cells were taken as a control.
   Post-treatment, the medium was aspirated, and the cells were washed
   with 1× phosphate-buffered saline (PBS) and treated with 20 μg/mL of PI
   for dead cells and 5 μg/mL of HOECHST as a counter nuclear stain in
   serum-free media for 15 min.^[197]75 For staining of the intracellular
   ROS productions, the cells were stained with DCFDA post-treatment with
   rotenone, Yashtimadhu alone, and Yashtimadhu choorna extract
   cotreatment. The cells were washed with 1× PBS and stained with 25 μM
   of DCFDA and 5 μg/mL of HOECHST in serum-free media and stained for 15
   min in the dark. Imaging was done with Zoe Imager, BioRad. The images
   were processed, and analysis of live–dead staining and ROS staining was
   measured using the ImageJ tool, NIH, USA.^[198]76 Cell viability was
   calculated with respect to untreated cells, and % ROS was calculated
   with respect to nuclear counterstained cells.

Immunoblotting Analysis

   IMR-32 cells were seeded at 3 × 10^4 cells/well in collagen-coated
   6-well plates and differentiated with retinoic acid. Rotenone (100 nM),
   Yashtimadhu choorna extract (200 μg/mL), and 100 nM of rotenone +
   Yashtimadhu choorna extract cotreatment was done for 48 h, while
   untreated cells were taken as a control. Post-treatment, the medium was
   removed, and the cells were washed with ice-cold 1× PBS. The cells were
   scraped and harvested in a lysis buffer containing 4% sodium dodecyl
   sulfate (SDS) in 50 mM triethylammonium bicarbonate (TEABC), with
   sodium orthovanadate (1 mM), sodium pyrophosphatase (2.5 mM), and
   beta-glycerophosphate (1 mM). The lysates were probe-sonicated on ice
   using Q-Sonica (Cole-Parmer, India) and heated at 95 °C on a dry bath
   for 10 min, followed by centrifugation at 12,000g for 20 min. The
   supernatant was aspirated into a new tube, and the protein
   concentration was estimated using the BCA protein estimation assay kit.

   Immunoblotting was performed as described previously.^[199]77 Briefly,
   an equal amount of protein was loaded across all treatments,
   electrophoretically resolved, and transferred onto the nitrocellulose
   membrane. The membranes were probed for the following proteins, with
   their dilution factors used for analysis, cleaved caspase-3 (1:1000),
   p-ERK1/2 (T202/Y204, 1:1000), TH (1:1000), SOD1 (1:1000), VDAC1
   (1:1000), AIFM1 (1:1000), PDHA1 (1:1000), CS (1:1000), and IDH1
   (1:1000), while β-actin (HRP-conjugated, 1:50,000) was used as a
   loading control. The blots were then incubated with the respective
   secondary antibodies (1:3000 dilution) and washed prior to imaging.
   Immunoreactive protein bands were visualized with Clarity ECL Substrate
   and captured onto X-ray films. The X-ray films were scanned, and
   densitometry analysis was carried out with ImageJ software, NIH,
   USA.^[200]76 The area under the curve was used to calculate protein
   expression, normalized with β-actin, and expressed as FC with respect
   to control.

Proteomics Sample Preparation

   IMR-32 cells were seeded at 1 × 10^5 cells/plate in 10 cm
   collagen-coated plates and differentiated with retinoic acid.
   Differentiated IMR-32 cells were treated with rotenone (100 nM),
   rotenone (100 nM) + Yashtimadhu choorna extract (200 μg/mL)
   cotreatment, and Yashtimadhu choorna extract (200 μg/mL) alone. The
   untreated cells were taken as a control. The cells were treated as
   independent biological duplicates and harvested. Briefly, the cells
   were washed thrice in ice-cold 1× PBS and scraped-off with lysis buffer
   [4% SDS in 50 mM TEABC with sodium pyrophosphate (2.5 mM), sodium
   orthovanadate (1 mM), and β-glycerophosphate (1 mM)], and the lysate
   was prepared as mentioned above. Protein concentration was estimated
   using the BCA assay, and the same was confirmed visually resolving on a
   10% SDS-polyacrylamide gel electrophoresis (PAGE) gel. Based on the
   protein concentrations, 300 μg of protein from each treatment was
   taken, reduced with dithiothreitol, and alkylated with iodoacetamide.
   The proteins were then precipitated with ice-cold acetone overnight at
   −20 °C. Precipitated proteins were reconstituted in 50 mM of TEABC and
   digested with TPCK-trypsin overnight at a ratio of 1:20 of
   enzyme/protein at 37 °C.^[201]32 Digestion efficiency was evaluated by
   resolving the samples on 10% SDS-PAGE. The peptides were dried
   overnight in SpeedVac and stored at −20 °C until labeling.

Tandem Mass Tag Labeling

   Peptide samples were reconstituted in 50 mM of TEABC, and their
   concentrations were estimated with the Pierce Peptide estimation kit.
   The peptide sample (50 μg) from each condition was used for labeling,
   with labels from the TMT 10plex kit. Peptides from the four different
   samples were labeled as follows: (i) untreated cells (C), (ii) 100 nM
   rotenone (R), (iii) 100 nM rotenone + 200 μg/mL Yashtimadhu choorna
   extract (RY), and (iv) 200 μg/mL Yashtimadhu choorna extract (Y), with
   four labels from the TMT 10plex kit, viz., 126 for C, 127N for R, 128C
   for RY, and 129N for Y. Both the biological replicates were
   independently labeled using the manufacturers’ protocol. The TMT labels
   were reconstituted in anhydrous acetonitrile (ACN) and vortexed for
   dissolution. The respective TMT tags were added to both the replicates
   and incubated at room temperature for 1 h and quenched by adding 8 μL
   of 5% hydroxylamine. The samples were pooled and dried overnight using
   SpeedVac. The dried samples were stored at −20 °C until peptide
   fractionation.

Peptide Fractionation and Clean-Up

   Peptide fractionation was carried out using a strong cation exchange
   (SCX) Stage Tip protocol as previously described.^[202]78 Briefly, the
   SCX material was stacked onto 200 μL tips and activated with 100% ACN,
   followed by equilibration with 2% trifluoroacetic acid (TFA). Peptides
   were reconstituted in 2% TFA and loaded onto the Stage Tips. The
   flow-through was passed twice, followed by washing with 0.2% TFA.
   Elution was carried out with different concentrations of ammonium
   acetate in ACN (50, 75, 125, 200, and 300 mM) into five separate
   fractions, and the sixth fraction was collected in 5% ammonium
   hydroxide with 80% ACN. The six fractions were collected in individual
   tubes and dried.

   Peptide desalting was carried out with the C-18 Stage Tip method, as
   described previously.^[203]79 The C-18 material was stacked onto 200 μL
   tips, activated with 100% ACN, and equilibrated with 0.1% formic acid.
   Fractionated peptide samples were reconstituted in 0.1% formic acid and
   loaded onto the C-18 material, and the flow-through was passed twice,
   followed by washing with 0.1% formic acid and elution with 40% ACN in
   0.1% formic acid. The dried fractions were stored at −20 °C until MS
   analysis.

LC–MS/MS Analysis

   LC–MS/MS analysis was carried out using an Orbitrap Fusion Tribrid mass
   spectrometer (Thermo Fisher Scientific, Bremen, Germany) coupled with
   an Easy-nl C1200 nanoflow UHPLC (Thermo Scientific, Odense, Denmark).
   The fractionated and dried peptides were reconstituted using 0.1%
   formic acid and introduced into the nanoViper trap column (75 μm × 2 cm
   and 3 μm, C18) (Thermo Fisher Scientific). Peptides were resolved on an
   EASY-Spray C18 Column (75 μm × 50 cm, 275 μm, 100 Å) maintained at a
   temperature of 40 °C. A gradient of 5–35% solvent B (0.1% formic acid
   in 80% ACN) at a flow rate of 300 nL/min for 110 min was used for
   peptide resolution. A total run time of 140 min, inclusive of the
   column conditioning and sample loading, was used for the LC–MS/MS
   analysis.

   The data-dependent acquisition was carried out within a mass range of
   400-1600 m/z in the Orbitrap mass analyzer at a resolution of 120,000
   at 200 m/z. Most intense precursor ions were selected for MS/MS
   fragmentation at a top speed data-dependent mode with a maximum cycle
   time of 3 s. Higher collision energy dissociation (HCD) fragmentation
   mode with a normalized collision energy of 35% was used in a scan range
   of 400–1600 m/z at a resolution of 60,000 at 200 m/z using the Orbitrap
   mass analyzer. Peptide charge states were set to 2–6, and a dynamic
   exclusion at 30 s, with a 10 ppm mass window, was used. Data
   acquisition was carried out in technical triplicates for both the
   biological replicates.

Database Search for Peptide and Protein Identification

   MS raw data files were processed using Proteome Discoverer, version 2.2
   (Thermo Fisher Scientific, Bremen, Germany). The data were searched
   against the human protein database RefSeq109 along with known
   contaminants (containing 81,096 entries and 116 contaminants), using
   SequestHT and MASCOT search algorithms. The search parameters were used
   as follows: a minimum peptide length of seven amino acids, with trypsin
   as the proteolytic enzyme and one missed cleavage. Precursor and
   fragment level mass tolerances were set at 10 ppm and 0.05 Da,
   respectively. TMT-modification at the peptide N-terminus and lysine
   residues and carbamidomethylation of cysteine were set as fixed
   modifications. Oxidation of methionine, protein N-terminal acetylation,
   was set as a dynamic modification. The percolator node in the consensus
   workflow was used to compute the false discovery rate (FDR), applied at
   1% at the peptide and PSM level. Data normalization was carried out on
   the total peptide amount using Proteome Discoverer.

Data Analysis

   The results file from Proteome Discoverer was used for further
   analysis. Perseus^[204]80 was used for PCA, computing the FC, and its
   respective logarithmic value at base-2 (log2 FC), which was used for
   calculating the p-value and Benjamini–Hochberg FDR-corrected p-value,
   that is, the q-value. Morpheus, Broad Institute
   ([205]https://software.broadinstitute.org/morpheus/), was used for
   generating heat maps. Sankey diagram was generated using an online
   Sankey generator
   ([206]http://sankey-diagram-generator.acquireprocure.com/). Proteins
   were classified and categorized based on Gene Ontology analysis using
   FunRich^[207]81 and pathway analysis using Enrichr
   ([208]https://amp.pharm.mssm.edu/Enrichr/)^[209]82 and Reactome^[210]83
   online tools. The proteins identified were also classified based on
   comparison with databases such as MitoCarta, version 2.0,^[211]84 The
   Autophagy Database,^[212]85 and the E3-ligases Database.^[213]63 PPI
   network analysis was carried out with STRING.^[214]86

Data Records

   The MS raw data and the Proteome Discoverer-searched data were
   submitted to the ProteomeXchange Consortium
   ([215]http://proteomecentral.proteomexchange.org) via the PRIDE
   repository^[216]87 with the data set identifier PXD019672.

Acknowledgments