Bhavneet
Kaur‡
ab,
Ajay
Bhat‡
ab,
Rahul
Chakraborty
ab,
Khushboo
Adlakha
a,
Shantanu
Sengupta
ab,
SoumyaSinha
Roy
*ab and
Kausik
Chakraborty
*ab
aGenomics and Molecular Medicine, CSIR-IGIB, Mathura Road, New Delhi, India. E-mail: kausik@igib.res.in; kausik@igib.in
bAcademy of Scientific & Innovative Research (AcSIR), CSIR-IGIB, Mathura Road Campus, New Delhi, India
First published on 19th January 2018
Perturbations affecting the homoeostasis of endoplasmic reticulum (ER) activate an adaptive signaling known as the unfolded protein response or UPR. Many studies have reported the association between neurological disorders and ER stress. Decreasing ER stress may therefore aid in therapeutic control of neuronal diseases. Sodium 4-phenylbutyrate (4-PBA), a small molecule, has been shown to alleviate ER stress and various neurological diseases, but the mechanistic basis of its action is not well understood. Using an iTRAQ based LC-MS technique we have delineated the effect of 4-PBA on the proteome of human neuroblastoma cells (SK-N-SH) during Tunicamycin-induced ER stress. The proteomic profile of 4-PBA-treated cells revealed that 4-PBA does not alter the cellular proteome to adapt towards ER stress. However, it can alleviate both the toxicity and proteomic alterations, induced by an ER stress inducer. Hence, the therapeutic effect of 4-PBA is primarily due to its ability to resolve ER stress rather than its ability to alter the expression of proteins required for maintaining ER proteostasis. Thus, we posit here that 4-PBA acts as an authentic chemical chaperone by aiding protein folding in the ER.
Chaperones play a key role during ER stress in preventing incomplete and unfolded proteins from forming aggregates and assisting in the proper folding of defective proteins in the ER lumen.15,16 There are two major types of chaperones: molecular and chemical chaperones. Molecular chaperones are proteins that help other proteins to acquire proper conformation by interacting with them. Hsp70, a classical example of molecular chaperones, functions by binding to unfolded polypeptides and prevents aggregation. It is known to play a role in reducing the levels of aggregated α-synuclein in both in vivo and in vitro models.17 Chemical chaperones are small molecular weight compounds which assist in folding and stability of proteins. These are two major types: osmotic (polyols, amino acids, amino acid derivatives and methylamines) and hydrophobic compounds (bile salts and sodium 4-phenylbutyrate).18,19 Sodium 4-phenylbutyrate (4-PBA) is a hydrophobic short chain fatty acid. It is an FDA-approved drug for the treatment of urea cycle disorders.20 4-PBA has additionally shown promising results in various diseases such as cancer, spinal muscular atrophy, cystic fibrosis, and neurodegenerative diseases associated with the folding of proteins, such as ALS (amyotrophic lateral sclerosis), Huntington's disease, Alzheimer's disease and Parkinson's disease.21–23 Many reports suggest that 4-PBA can act as a chemical chaperone by preventing aggregation of misfolded proteins.24,25 A competing model proposes that 4-PBA has an HDAC (Histone deacetylase) inhibitor activity, and thereby may regulate the expression of various neuronal genes during neurodegenerative disorders which involve aberrant histone acetylation.23,26,27 However, despite the fact that 4-PBA has therapeutic potential in proteostasis disorders, the validity of the competing proposals has not been authenticated.
In this study using a human neuroblastoma cell line as a model system, we investigated the effect of 4-PBA during Tm (prevents N-linked glycosylation) induced ER stress. With the help of iTRAQ based quantitative proteomics, we identified the pathways affected by 4-PBA. This report highlights the first proteomic profile of 4-PBA-treated neuronal cells during ER stress. We show that the most likely route of 4-PBA action is through the alleviation of ER stress itself and not through the modulation of stress-response genes.
LDH assay was performed using a cytotoxicity detection kit (cytotoxicity detection kit plus, Roche). In this assay, cells were grown in 96-well flat bottom plates at a seeding density of 1 × 104 cells per well, and upon 70–80% confluency, cells were treated with Tm and 4-PBA. Following treatment, LDH assay was performed according to the manufacturer's protocol. Briefly, 100 μl of freshly prepared reaction mix was added to an equal volume of the supernatant collected from each well for every experimental group and was incubated for 30 min at room temperature. After incubation, absorbance was measured at 490 nm using a microplate reader. Further, relative cytotoxicity was calculated using the following equation: {(Ab_Exp − Ab_Con)/(Ab_Pc − Ab_Con)} × 100, where Ab_Exp = absorbance of the experimental group, Ab_Con = absorbance of the control group, and Ab_Pc = absorbance of the positive control (provided with the kit).
To have a comprehensive understanding of the protective effect of 4-PBA during ER stress in neuronal cells, we performed 4-plex iTRAQ-based relative quantitative proteomics experiment under different conditions: control (without any treatment), Tm treated, 4-PBA treated and a combination of 4-PBA and Tm treatment. In the three biological replicates, we identified 3665, 3199, and 3510 proteins respectively, with 1% FDR and at least two unique peptides. Of these, 2023 proteins were identified in all the three experiments (Fig. 2 and ESI,† Table S1). The % CV was calculated across the three biological replicates to check the reproducibility of the experiments. Out of 2023 proteins, 88% of the proteins had a CV of <20% (data not shown). The relative expression of GRP78 (a well-known marker of ER stress) acquired from this experiment was in agreement with the fold change obtained from the immunoblotting technique (ESI,† Fig. S2). This validated the relative quantification acquired through the iTRAQ approach.
Fig. 2 Venn diagram displaying the overlap of proteins identified in three biological replicates of iTRAQ experiments. |
We further investigated the differential expression of proteins in Tm-treated cells to identify the cellular response towards an ER stress inducer. Proteins were considered to be upregulated by Tm, if the fold change with respect to the control was ≥1.2 in all biological replicates. Similarly, a fold change of ≤0.8 was used for downregulated proteins. Using these criteria of selection, 101 proteins were found to be differentially expressed of which 84 were upregulated and 17 were downregulated (Table 1). In agreement with a previous report, Tm-treatment led to the overexpression of proteins involved in protein folding and cellular redox homeostasis.32 Several proteins like GRP78, DNAJB11, PDIA4, PDIA3, and ERO1 that are involved in unfolded protein response were upregulated. This further indicates that in our experimental conditions, Tm was able to cause ER stress and induce unfolded protein response. These differentially expressed proteins were classified based on their Gene Ontology (GO) parameters of the biological function and cellular localization using the Database for Annotation, Visualization and Integrated Discovery (DAVID). GO analyses based on cellular localization revealed that the majority of the Tm-induced differentially expressed proteins are localized in the mitochondria, extracellular exosomes and endoplasmic reticulum (Fig. 3 and ESI,† Table S1). Surprisingly 54% of the differentially expressed proteins belonged to extracellular exosomes, highlighting a yet underappreciated connection that demands further investigation to understand the modulation of extracellular communication during ER stress. The biological function analysis further showed that proteins involved in mitochondrial ATP synthesis coupled proton transport, protein folding, response to ER stress, cellular redox homeostasis, and nucleosome assembly were also significantly enriched (Fig. 3).
Up-regulated | Fold change: Tm/control | |||
---|---|---|---|---|
Acession ID | Description | Rep 1 | Rep 2 | Rep 3 |
P11021 | 78 kDa glucose-regulated protein OS = Homo sapiens GN = HSPA5 PE = 1 SV = 2 | 3.47 | 3.17 | 3.09 |
P30101 | Protein disulfide-isomerase A3 OS = Homo sapiens GN = PDIA3 PE = 1 SV = 4 | 1.91 | 1.94 | 1.85 |
P13667 | Protein disulfide-isomerase A4 OS = Homo sapiens GN = PDIA4 PE = 1 SV = 2 | 2.3 | 2.27 | 2.11 |
P07237 | Protein disulfide-isomerase OS = Homo sapiens GN = P4HB PE = 1 SV = 3 | 1.82 | 1.87 | 1.75 |
P06576 | ATP synthase subunit beta, mitochondrial OS = Homo sapiens GN = ATP5B PE = 1 SV = 3 | 1.42 | 1.39 | 1.4 |
P25705 | ATP synthase subunit alpha, mitochondrial OS = Homo sapiens GN = ATP5A1 PE = 1 SV = 1 | 1.37 | 1.32 | 1.36 |
B4DGP8 | Calnexin OS = Homo sapiens GN = CANX PE = 2 SV = 1 | 1.86 | 1.92 | 1.82 |
H6VRG3 | Keratin 1 OS = Homo sapiens GN = KRT1 PE = 3 SV = 1 | 1.52 | 6.05 | 1.6 |
O15240 | Neurosecretory protein VGF OS = Homo sapiens GN = VGF PE = 1 SV = 2 | 1.24 | 1.25 | 1.31 |
Q6IAW5 | CALU protein OS = Homo sapiens GN = CALU PE = 2 SV = 1 | 1.49 | 1.73 | 1.57 |
P50454 | Serpin H1 OS = Homo sapiens GN = SERPINH1 PE = 1 SV = 2 | 1.45 | 1.49 | 1.4 |
K7ELL7 | Glucosidase 2 subunit beta OS = Homo sapiens GN = PRKCSH PE = 4 SV = 1 | 1.54 | 1.48 | 1.51 |
P08758 | Annexin A5 OS = Homo sapiens GN = ANXA5 PE = 1 SV = 2 | 1.44 | 1.4 | 1.34 |
P07355 | Annexin A2 OS = Homo sapiens GN = ANXA2 PE = 1 SV = 2 | 1.5 | 1.43 | 1.44 |
G3XAI2 | Laminin subunit beta-1 OS = Homo sapiens GN = LAMB1 PE = 2 SV = 1 | 1.4 | 1.4 | 1.32 |
A8K401 | Prohibitin, isoform CRA_a OS = Homo sapiens GN = PHB PE = 2 SV = 1 | 1.5 | 1.37 | 1.45 |
J3KPX7 | Prohibitin-2 OS = Homo sapiens GN = PHB2 PE = 4 SV = 1 | 1.45 | 1.34 | 1.4 |
Q5TZZ9 | Annexin OS = Homo sapiens GN = ANXA1 PE = 2 SV = 1 | 1.41 | 1.47 | 1.41 |
P31930 | Cytochrome b–c 1 complex subunit 1, mitochondrial OS = Homo sapiens GN = UQCRC1 PE = 1 SV = 3 | 1.22 | 1.25 | 1.27 |
Q9BZQ8 | Protein Niban OS = Homo sapiens GN = FAM129A PE = 1 SV = 1 | 1.22 | 1.32 | 1.28 |
P05141 | ADP/ATP translocase 2 OS = Homo sapiens GN = SLC25A5 PE = 1 SV = 7 | 1.33 | 1.31 | 1.26 |
Q96A33 | Coiled-coil domain-containing protein 47 OS = Homo sapiens GN = CCDC47 PE = 1 SV = 1 | 1.34 | 1.22 | 1.2 |
Q8NCF7 | cDNA FLJ90278 fis, clone NT2RP1000325, highly similar to phosphate carrier protein, mitochondrial precursor OS = Homo sapiens PE = 2 SV = 1 | 1.32 | 1.34 | 1.32 |
P23284 | Peptidyl-prolyl cis–trans isomerase B OS = Homo sapiens GN = PPIB PE = 1 SV = 2 | 1.97 | 2.22 | 1.92 |
P35908 | Keratin, type II cytoskeletal 2 epidermal OS = Homo sapiens GN = KRT2 PE = 1 SV = 2 | 1.65 | 1.59 | 1.56 |
J3KPF3 | 4F2 cell-surface antigen heavy chain OS = Homo sapiens GN = SLC3A2 PE = 4 SV = 1 | 1.45 | 1.46 | 1.43 |
P13521 | Secretogranin-2 OS = Homo sapiens GN = SCG2 PE = 1 SV = 2 | 1.29 | 1.25 | 1.28 |
Q5U0D2 | Putative uncharacterized protein DKFZp686P11128 OS = Homo sapiens GN = TAGLN PE = 2 SV = 1 | 1.39 | 1.42 | 1.35 |
A8K878 | cDNA FLJ77177, highly similar to Homo sapiens arginine-rich, mutated in early stage tumors (ARMET), mRNA OS = Homo sapiens PE = 2 SV = 1 | 2 | 1.85 | 1.87 |
P30040 | Endoplasmic reticulum resident protein 29 OS = Homo sapiens GN = ERP29 PE = 1 SV = 4 | 1.41 | 1.36 | 1.42 |
Q9BS26 | Endoplasmic reticulum resident protein 44 OS = Homo sapiens GN = ERP44 PE = 1 SV = 1 | 1.49 | 1.51 | 1.34 |
Q96HE7 | ERO1-like protein alpha OS = Homo sapiens GN = ERO1L PE = 1 SV = 2 | 1.39 | 1.39 | 1.37 |
Q15293 | Reticulocalbin-1 OS = Homo sapiens GN = RCN1 PE = 1 SV = 1 | 1.56 | 1.51 | 1.44 |
Q9BRK5 | 45 kDa calcium-binding protein OS = Homo sapiens GN = SDF4 PE = 1 SV = 1 | 1.31 | 1.21 | 1.31 |
P16401 | Histone H1.5 OS = Homo sapiens GN = HIST1H1B PE = 1 SV = 3 | 1.77 | 2.18 | 1.83 |
Q9UBS4 | DnaJ homolog subfamily B member 11 OS = Homo sapiens GN = DNAJB11 PE = 1 SV = 1 | 1.51 | 1.5 | 1.52 |
Q08ET0 | Cell proliferation-inducing protein 47 OS = Homo sapiens GN = hCG_39985 PE = 2 SV = 1 | 1.29 | 1.48 | 1.21 |
Q8TAS0 | ATP synthase subunit gamma (fragment) OS = Homo sapiens PE = 2 SV = 1 | 1.27 | 1.36 | 1.39 |
P48047 | ATP synthase subunit O, mitochondrial OS = Homo sapiens GN = ATP5O PE = 1 SV = 1 | 1.39 | 1.3 | 1.33 |
Q53GF9 | Full-length cDNA 5-PRIME end of clone CS0DF013YM24 of fetal brain of Homo sapiens (human) variant (fragment) OS = Homo sapiens PE = 2 SV = 1 | 1.58 | 1.53 | 1.43 |
P80303 | Nucleobindin-2 OS = Homo sapiens GN = NUCB2 PE = 1 SV = 2 | 1.53 | 1.32 | 1.32 |
Q13162 | Peroxiredoxin-4 OS = Homo sapiens GN = PRDX4 PE = 1 SV = 1 | 1.24 | 1.28 | 1.24 |
O75947 | ATP synthase subunit d, mitochondrial OS = Homo sapiens GN = ATP5H PE = 1 SV = 3 | 1.29 | 1.31 | 1.31 |
P20674 | Cytochrome c oxidase subunit 5A, mitochondrial OS = Homo sapiens GN = COX5A PE = 1 SV = 2 | 1.33 | 1.42 | 1.22 |
Q5T0G8 | Annexin OS = Homo sapiens GN = ANXA11 PE = 2 SV = 1 | 1.4 | 1.36 | 1.48 |
Q14696 | LDLR chaperone MESD OS = Homo sapiens GN = MESDC2 PE = 1 SV = 2 | 1.22 | 1.63 | 1.36 |
Q96JZ5 | SM-11044 binding protein, isoform CRA_b OS = Homo sapiens GN = SMBP PE = 2 SV = 1 | 1.42 | 1.21 | 1.27 |
B4DR61 | Protein transport protein Sec61 subunit alpha isoform 1 OS = Homo sapiens GN = SEC61A1 PE = 2 SV = 1 | 1.75 | 2.04 | 1.71 |
Q53XJ5 | Peptidyl-prolyl cis–trans isomerase OS = Homo sapiens GN = FKBP2 PE = 2 SV = 1 | 1.39 | 1.44 | 1.37 |
B7Z5L4 | cDNA FLJ61340, highly similar to Homo sapiens seizure related 6 homolog-like 2 (SEZ6L2), transcript variant 2, mRNA OS = Homo sapiens PE = 2 SV = 1 | 1.62 | 1.72 | 1.76 |
Q5T8U7 | Surfeit 4 OS = Homo sapiens GN = SURF4 PE = 2 SV = 1 | 1.57 | 1.26 | 1.43 |
Q8TCT9 | Minor histocompatibility antigen H13 OS = Homo sapiens GN = HM13 PE = 1 SV = 1 | 1.54 | 1.2 | 1.25 |
P51970 | NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 8 OS = Homo sapiens GN = NDUFA8 PE = 1 SV = 3 | 1.34 | 1.39 | 1.27 |
Q6FHT8 | RNP24 protein OS = Homo sapiens GN = RNP24 PE = 2 SV = 1 | 1.37 | 1.37 | 1.26 |
O75367 | Core histone macro-H2A.1 OS = Homo sapiens GN = H2AFY PE = 1 SV = 4 | 1.79 | 1.85 | 1.72 |
B4DVE1 | cDNA FLJ53478, highly similar to Galectin-3-binding protein OS = Homo sapiens PE = 2 SV = 1 | 1.37 | 1.39 | 1.25 |
O15173 | Membrane-associated progesterone receptor component 2 OS = Homo sapiens GN = PGRMC2 PE = 1 SV = 1 | 1.32 | 1.34 | 1.29 |
Q16222 | UDP-N-acetylhexosamine pyrophosphorylase OS = Homo sapiens GN = UAP1 PE = 1 SV = 3 | 1.68 | 1.58 | 1.4 |
A8KA82 | DnaJ (Hsp40) homolog, subfamily C, member 3 OS = Homo sapiens GN = DNAJC3 PE = 2 SV = 1 | 1.51 | 1.52 | 1.39 |
Q9BRR6 | ADP-dependent glucokinase OS = Homo sapiens GN = ADPGK PE = 1 SV = 1 | 1.32 | 1.43 | 1.22 |
P31949 | Protein S100-A11 OS = Homo sapiens GN = S100A11 PE = 1 SV = 2 | 1.5 | 1.44 | 1.47 |
Q8NI22 | Multiple coagulation factor deficiency protein 2 OS = Homo sapiens GN = MCFD2 PE = 1 SV = 1 | 1.33 | 1.23 | 1.2 |
P30049 | ATP synthase subunit delta, mitochondrial OS = Homo sapiens GN = ATP5D PE = 1 SV = 2 | 1.3 | 1.61 | 1.62 |
Q9NZ45 | CDGSH iron–sulfur domain-containing protein 1 OS = Homo sapiens GN = CISD1 PE = 1 SV = 1 | 1.32 | 1.3 | 1.27 |
E9PN17 | ATP synthase subunit g, mitochondrial OS = Homo sapiens GN = ATP5L PE = 2 SV = 1 | 1.45 | 1.42 | 1.44 |
Q9Y3A6 | Transmembrane emp24 domain-containing protein 5 OS = Homo sapiens GN = TMED5 PE = 1 SV = 1 | 1.59 | 1.63 | 1.73 |
P07305 | Histone H1.0 OS = Homo sapiens GN = H1F0 PE = 1 SV = 3 | 2.34 | 1.73 | 1.99 |
P35610 | Sterol O-acyltransferase 1 OS = Homo sapiens GN = SOAT1 PE = 1 SV = 3 | 1.21 | 1.38 | 1.35 |
H0Y886 | NADH dehydrogenase [ubiquinone] 1 beta subcomplex subunit 5, mitochondrial (fragment) OS = Homo sapiens GN = NDUFB5 PE = 4 SV = 1 | 1.57 | 1.8 | 1.3 |
Q9NYB0 | Telomeric repeat-binding factor 2-interacting protein 1 OS = Homo sapiens GN = TERF2IP PE = 1 SV = 1 | 1.27 | 1.66 | 2.73 |
G3V325 | Pentatricopeptide repeat-containing protein 1, mitochondrial OS = Homo sapiens GN = ATP5J2-PTCD1 PE = 4 SV = 1 | 1.47 | 1.28 | 1.43 |
A8K0F7 | cDNA FLJ76587, highly similar to Homo sapiens vitamin K epoxide reductase complex, subunit 1-like 1 (VKORC1L1), mRNA OS = Homo sapiens PE = 2 SV = 1 | 1.33 | 1.36 | 1.24 |
B2R8A2 | cDNA, FLJ93804, highly similar to Homo sapiens gp25L2 protein (HSGP25L2G), mRNA OS = Homo sapiens PE = 2 SV = 1 | 1.41 | 1.53 | 1.45 |
Q6IBU4 | SDF2 protein OS = Homo sapiens GN = SDF2 PE = 2 SV = 1 | 1.36 | 1.23 | 1.68 |
Q71UI9 | Histone H2A.V OS = Homo sapiens GN = H2AFV PE = 1 SV = 3 | 2.77 | 2.62 | 2.34 |
Q6IAM7 | SPC18 protein OS = Homo sapiens GN = SPC18 PE = 2 SV = 1 | 1.69 | 1.21 | 1.35 |
Q13425 | Beta-2-syntrophin OS = Homo sapiens GN = SNTB2 PE = 1 SV = 1 | 1.37 | 1.49 | 1.21 |
Q9UDW1 | Cytochrome b–c 1 complex subunit 9 OS = Homo sapiens GN = UQCR10 PE = 1 SV = 3 | 1.58 | 1.41 | 1.58 |
P05204 | Non-histone chromosomal protein HMG-17 OS = Homo sapiens GN = HMGN2 PE = 1 SV = 3 | 1.76 | 4.21 | 1.29 |
P68431 | Histone H3.1 OS = Homo sapiens GN = HIST1H3A PE = 1 SV = 2 | 6.1 | 3.9 | 9.3 |
A8K2Q6 | Peptidyl-prolyl cis–trans isomerase OS = Homo sapiens PE = 2 SV = 1 | 1.57 | 1.37 | 1.37 |
Q6I9V5 | SLC25A6 protein OS = Homo sapiens GN = SLC25A6 PE = 2 SV = 1 | 1.33 | 1.54 | 1.35 |
Q5U0C3 | RAP1A, member of RAS oncogene family OS = Homo sapiens PE = 2 SV = 1 | 1.25 | 1.49 | 1.25 |
Q71DI3 | Histone H3.2 OS = Homo sapiens GN = HIST2H3A PE = 1 SV = 3 | 5.06 | 4.58 | 4.16 |
Down regulated | Fold change: Tm/control | |||
---|---|---|---|---|
Acession ID | Description | Rep 1 | Rep 2 | Rep 3 |
J3KTA4 | Probable ATP-dependent RNA helicase DDX5 OS = Homo sapiens GN = DDX5 PE = 3 SV = 1 | 0.67 | 0.69 | 0.71 |
Q14566 | DNA replication licensing factor MCM6 OS = Homo sapiens GN = MCM6 PE = 1 SV = 1 | 0.75 | 0.77 | 0.74 |
P31689 | DnaJ homolog subfamily A member 1 OS = Homo sapiens GN = DNAJA1 PE = 1 SV = 2 | 0.76 | 0.73 | 0.77 |
Q9UNF1 | Melanoma-associated antigen D2 OS = Homo sapiens GN = MAGED2 PE = 1 SV = 2 | 0.8 | 0.76 | 0.66 |
Q96T88 | E3 ubiquitin-protein ligase UHRF1 OS = Homo sapiens GN = UHRF1 PE = 1 SV = 1 | 0.74 | 0.75 | 0.77 |
Q8IV08 | Phospholipase D3 OS = Homo sapiens GN = PLD3 PE = 1 SV = 1 | 0.69 | 0.67 | 0.74 |
Q9Y5L4 | Mitochondrial import inner membrane translocase subunit Tim13 OS = Homo sapiens GN = TIMM13 PE = 1 SV = 1 | 0.76 | 0.72 | 0.79 |
Q7Z7L8 | Uncharacterized protein C11orf96 OS = Homo sapiens GN = C11orf96 PE = 1 SV = 3 | 0.76 | 0.61 | 0.53 |
Q92558 | Wiskott–Aldrich syndrome protein family member 1 OS = Homo sapiens GN = WASF1 PE = 1 SV = 1 | 0.79 | 0.64 | 0.78 |
O75243 | R30783_1 OS = Homo sapiens PE = 4 SV = 1 | 0.69 | 0.68 | 0.8 |
Q7Z7K6 | Centromere protein V OS = Homo sapiens GN = CENPV PE = 1 SV = 1 | 0.8 | 0.44 | 0.28 |
Q2L6I0 | FB19 protein OS = Homo sapiens GN = PPP1R10 PE = 2 SV = 1 | 0.8 | 0.8 | 0.78 |
B2R4N3 | cDNA, FLJ92155, highly similar to Homo sapiens ubiquitin-like 5 (UBL5), mRNA OS = Homo sapiens PE = 4 SV = 1 | 0.74 | 0.61 | 0.5 |
Q9Y6H1 | Coiled-coil-helix–coiled-coil-helix domain-containing protein 2, mitochondrial OS = Homo sapiens GN = CHCHD2 PE = 1 SV = 1 | 0.76 | 0.58 | 0.76 |
Q59EN5 | Prosaposin variant (fragment) OS = Homo sapiens PE = 2 SV = 1 | 0.75 | 0.51 | 0.5 |
A9CQZ4 | Dihydropyrimidinase-like 2 long form (fragment) OS = Homo sapiens GN = DPYSL2 PE = 2 SV = 1 | 0.71 | 0.67 | 0.77 |
Q9UMZ1 | Prothymosin a14 OS = Homo sapiens PE = 1 SV = 1 | 0.72 | 0.77 | 0.48 |
Fig. 3 Gene ontology (GO) analysis of Tm induced differentially expressed proteins. (i) Enriched biological functions, (ii) enriched cellular compartment. |
4-PBA is known to alleviate ER stress induced by Tm. We thus asked if 4-PBA could upregulate the protein quality control machinery of ER to help survival during ER stress. We then assessed the proteins that are modulated by 4-PBA alone (in the absence of Tm). We found that 4-PBA-treatment did not alter the proteome significantly as only one protein, Tmed5 – Transmembrane emp24 domain-containing protein, was up-regulated and two proteins, Osgep-(Probable tRNA threonylcarbamoyladenosine biosynthesis protein) and Tspo (Putative peripheral benzodiazepine receptor-related protein), were downregulated. These proteins that are differentially expressed upon treatment with 4-PBA are not bonafide members of the protein quality control machinery in the ER. Thus, it is apparent that 4-PBA treatment does not alter the expression of proteins required for maintaining ER homeostasis.
To check if addition of 4-PBA had an effect on the expression of proteins that were modulated by Tm, we performed a Jack-knife resampling analysis30 to investigate the significance of changes induced by 4-PBA during ER stress. For this analysis, proteins that were differentially expressed by Tm treatment in one replicate (fold change ≥1.2 for upregulated proteins, ≤0.8 for downregulated proteins) were taken and the average ratio of (4-PBA + Tm)/control and 4-PBA/control for these proteins in the other two replicates was calculated. This was repeated for all the replicates using the first, second and third replicate as a reference point and a box plot of the fold change was plotted (Fig. 4). In all the three replicates the average ratio of Tm/control was significantly different from (4-PBA + Tm)/control for both up- and downregulated proteins during Tm-treatment. This indicates that on average the expression of proteins induced or repressed by Tm could be reverted to near control levels. Among the Tm-induced differentially expressed proteins, a list of proteins whose expression was significantly reverted by 4-PBA was identified by considering proteins whose fold change for (4-PBA + Tm)/Tm is ≥1.1 or ≤0.9 with p-value ≤0.05 (Table 2 and Fig. 5). Most of the pathways altered by Tm were recovered by 4-PBA (Fig. 6). Both ER quality control and mitochondrial respiration-related proteins were suppressed by 4-PBA during ER stress. Thus the study demonstrates that 4-PBA has a global protective effect on proteomic alterations induced by Tm.
Acession ID | Description | Fold change (Tm + 4-PBA)/Tm | p-Value | Status in Tm treatment |
---|---|---|---|---|
P11021 | 78 kDa glucose-regulated protein OS = Homo sapiens GN = HSPA5 PE = 1 SV = 2 | 0.8 | 3.31 × 10−3 | UP |
P30101 | Protein disulfide-isomerase A3 OS = Homo sapiens GN = PDIA3 PE = 1 SV = 4 | 0.82 | 2.75 × 10−3 | UP |
P13667 | Protein disulfide-isomerase A4 OS = Homo sapiens GN = PDIA4 PE = 1 SV = 2 | 0.82 | 1.47 × 10−3 | UP |
P07237 | Protein disulfide-isomerase OS = Homo sapiens GN = P4HB PE = 1 SV = 3 | 0.86 | 1.41 × 10−3 | UP |
P06576 | ATP synthase subunit beta, mitochondrial OS = Homo sapiens GN = ATP5B PE = 1 SV = 3 | 0.89 | 2.30 × 10−2 | UP |
B4DGP8 | Calnexin OS = Homo sapiens GN = CANX PE = 2 SV = 1 | 0.86 | 4.80 × 10−3 | UP |
Q6IAW5 | CALU protein OS = Homo sapiens GN = CALU PE = 2 SV = 1 | 0.89 | 4.91 × 10−2 | UP |
K7ELL7 | Glucosidase 2 subunit beta OS = Homo sapiens GN = PRKCSH PE = 4 SV = 1 | 0.86 | 1.37 × 10−2 | UP |
A8K401 | Prohibitin, isoform CRA_a OS = Homo sapiens GN = PHB PE = 2 SV = 1 | 0.86 | 5.85 × 10−3 | UP |
J3KPX7 | Prohibitin-2 OS = Homo sapiens GN = PHB2 PE = 4 SV = 1 | 0.89 | 2.94 × 10−2 | UP |
P05141 | ADP/ATP translocase 2 OS = Homo sapiens GN = SLC25A5 PE = 1 SV = 7 | 0.9 | 3.42 × 10−3 | UP |
P23284 | Peptidyl-prolyl cis–trans isomerase BOS = Homo sapiens GN = PPIB PE = 1 SV = 2 | 0.82 | 2.61 × 10−2 | UP |
A8K878 | cDNA FLJ77177, highly similar to Homo sapiens arginine-rich, mutated in early stage tumors (ARMET), mRNA OS = Homo sapiens PE = 2 SV = 1 | 0.82 | 2.22 × 10−2 | UP |
P30040 | Endoplasmic reticulum resident protein 29 OS = Homo sapiens GN = ERP29 PE = 1 SV = 4 | 0.88 | 4.95 × 10−3 | UP |
Q96HE7 | ERO1-like protein alpha OS = Homo sapiens GN = ERO1L PE = 1 SV = 2 | 0.89 | 2.91 × 10−3 | UP |
Q15293 | Reticulocalbin-1 OS = Homo sapiens GN = RCN1 PE = 1 SV = 1 | 0.9 | 4.65 × 10−2 | UP |
Q8TAS0 | ATP synthase subunit gamma (fragment) OS = Homo sapiens PE = 2 SV = 1 | 0.84 | 1.17 × 10−2 | UP |
P48047 | ATP synthase subunit O, mitochondrial OS = Homo sapiens GN = ATP5O PE = 1 SV = 1 | 0.88 | 1.75 × 10−2 | UP |
Q53GF9 | Full-length cDNA 5-PRIME end of clone CS0DF013YM24 of fetal brain of Homo sapiens (human) variant (fragment) OS = Homo sapiens PE = 2 SV = 1 | 0.9 | 2.68 × 10−2 | UP |
Q13162 | Peroxiredoxin-4 OS = Homo sapiens GN = PRDX4 PE = 1 SV = 1 | 0.9 | 2.68 × 10−2 | UP |
P20674 | Cytochrome c oxidase subunit 5A, mitochondrial OS = Homo sapiens GN = COX5A PE = 1 SV = 2 | 0.83 | 2.16 × 10−2 | UP |
B4DR61 | Protein transport protein Sec61 subunit alpha isoform 1 OS = Homo sapiens GN = SEC61A1 PE = 2 SV = 1 | 0.72 | 3.45 × 10−2 | UP |
Q53XJ5 | Peptidyl-prolyl cis–trans isomerase OS = Homo sapiens GN = FKBP2 PE = 2 SV = 1 | 0.88 | 1.40 × 10−2 | UP |
P51970 | NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 8 OS = Homo sapiens GN = NDUFA8 PE = 1 SV = 3 | 0.86 | 3.50 × 10−3 | UP |
O75367 | Core histone macro-H2A.1 OS = Homo sapiens GN = H2AFY PE = 1 SV = 4 | 0.74 | 7.97 × 10−3 | UP |
O15173 | Membrane-associated progesterone receptor component 2 OS = Homo sapiens GN = PGRM C2 PE = 1 SV = 1 | 0.86 | 3.03 × 10−2 | UP |
P30049 | ATP synthase subunit delta, mitochondrial OS = Homo sapiens GN = ATP5D PE = 1 SV = 2 | 0.81 | 2.47 × 10−2 | UP |
E9PN17 | ATP synthase subunit g, mitochondrial OS = Homo sapiens GN = ATP5L PE = 2 SV = 1 | 0.88 | 2.61 × 10−2 | UP |
B2R8A2 | cDNA, FLJ93804, highly similar to Homo sapiens sgp25L2 protein (HSGP25L2G), mRNA OS = Homo sapiens PE = 2 SV = 1 | 0.89 | 3.67 × 10−2 | UP |
Q71UI9 | Histone H2A.V OS = Homo sapiens GN = H2AFV PE = 1 SV = 3 | 0.68 | 2.21 × 10−3 | UP |
Q71DI3 | Histone H3.2 OS = Homo sapiens GN = HIST2H3A PE = 1 SV = 3 | 0.62 | 1.45 × 10−3 | UP |
J3KTA4 | Probable ATP-dependent RNA helicase DDX5 OS = Homo sapiens GN = DDX5 PE = 3 SV = 1 | 1.14 | 1.03 × 10−2 | Down |
Q14566 | DNA replication licensing factor MCM6 OS = Homo sapiens GN = MCM6 PE = 1 SV = 1 | 1.1 | 2.26 × 10−2 | Down |
P31689 | DnaJ homolog subfamily A member 1 OS = Homo sapiens GN = DNAJA1 PE = 1 SV = 2 | 1.11 | 4.24 × 10−2 | Down |
Q9UNF1 | Melanoma-associated antigen D2 OS = Homo sapiens GN = MAGED2 PE = 1 SV = 2 | 1.19 | 2.67 × 10−2 | Down |
Q96T88 | E3 ubiquitin-protein ligase UHRF 1 OS = Homo sapiens GN = UHRF1 PE = 1 SV = 1 | 1.2 | 2.71 × 10−4 | Down |
Q8IV08 | Phospholipase D3 OS = Homo sapiens GN = PLD3 PE = 1 SV = 1 | 1.2 | 3.64 × 10−2 | Down |
O75243 | R30783_1 OS = Homo sapiens PE = 4 SV = 1 | 1.39 | 7.94 × 10−3 | Down |
Q7Z7K6 | Centromere protein V OS = Homo sapiens GN = CENPV PE = 1 SV = 1 | 2.39 | 3.17 × 10−2 | Down |
Q2L6I0 | FB19 protein OS = Homo sapiens GN = PPP1R10 PE = 2 SV = 1 | 1.22 | 6.04 × 10−3 | Down |
Tm is a well-known ER stress inducer; it inhibits N-linked glycosylation and leads to accumulation of unfolded glycoproteins in the ER, causing ER stress38,39 The proteomic profile of Tm treated neuronal cells is in agreement with previous reports. We found enhanced accumulation of proteins involved in protein folding and cellular redox homeostasis.32 Various studies have indicated the cross-talk between ER and mitochondria under stress conditions.28,40,41 In the present study, we found that most of the Tm-induced differentially expressed proteins localize in the ER or mitochondria. Enrichment of a large number of mitochondria or ER resident proteins further strengthens the known crosstalk between the two compartments during ER stress.28,40,41 The ER quality control machinery depends upon metabolic energy for proper folding and clearance of misfolded proteins.42,43 Bravo et al. demonstrated that there is an increase in mitochondrial respiration during ER stress. This was found to be an adaptive response.44 When ER stress is not resolved it leads to cell death by inducing mitochondrial dysfunction, and hence, the enrichment of mitochondrial proteins involved in energy synthesis, in our data, underlines the role of mitochondrial respiration in ER stress. However, this does not exclude a more complex and direct cross-talk between ER and mitochondria.
4-PBA reduces Tm-induced cell death and decreases the expression of UPR markers (GRP78 and CHOP). 4-PBA alone does not alter the quality control machinery of ER but in the presence of an ER stress inducer, it restores the altered ER stress induced expression of proteins towards unstressed levels. Most of the pathways affected by Tm were recovered by 4-PBA. 4-PBA suppresses the expression of UPR genes and toxicity induced by Tm and thus directs towards the possibility that 4-PBA works by decreasing ER stress instead of preconditioning the ER to cope better with stress. 4-PBA not only decreases the expression of UPR genes, but also recovers the expression of proteins involved in mitochondrial ATP synthesis. ER stress-induced cell death involves increased mitochondrial respiration, followed by apoptosis.44 Our study reveals that 4-PBA recovers the Tm-induced upregulation of proteins involved in both ER stress and mitochondrial respiration.
4-PBA has an HDAC (Histone deacetylase) inhibitor activity. It thus has the ability to alter the expression of genes which involve aberrant histone acetylation during neurological disorders.23,26,27 In a previous report by Mimori et al.,45 it was shown using structural analogs of 4-PBA that protection was indeed correlated with the in vitro chaperoning activity of the molecule and not HDAC7 binding activity. However, it could not exclude if 4-PBA activated other pathways to protect cells against UPR. Using a global measure of cellular response we support the view proposed by Mimori et al. that 4-PBA indeed does not have a protective effect by upregulating alternate protective pathways to combat ER stress. Its activity most likely is an outcome of its chaperoning activity.
In conclusion, our study demonstrates the first proteomic profile of 4-PBA during Tm treatment in human neuroblastoma cells. This study illustrates that 4-PBA exhibits a global recovery from the proteomic alterations induced by Tm but does not alter the cellular proteome to adapt towards ER stress. This supports the suspected role of 4-PBA as a bonafide chemical chaperone and suggests that 4-PBA may aid in protein folding of ER resident proteins to alleviate ER proteotoxicity.
4-PBA | Sodium 4-phenylbutyrate |
ALS | Amyotrophic lateral sclerosis |
ATCC | American Type Culture Collection |
ATF4 | Activating transcription factor 4 |
ATF6 | Activating transcription factor 6 |
ACN | Acetonitrile |
CHOP | CCAAT-enhancer-binding protein homologous protein |
% CV | Percentage coefficient of variation |
DAVID | Database for Annotation, Visualization and Integrated Discovery |
DMEM | Dulbecco's modified Eagle's medium |
ER | Endoplasmic reticulum |
FBS | Fetal bovine serum |
FDA | Food and Drug Administration |
FDR | False discovery rate |
GO | Gene ontology |
GRP78 | Glucose-regulated protein 78 |
HDAC | Histone deacetylase |
HRP | Horseradish peroxidase |
IAA | Iodoacetamide |
IDA | Information dependent acquisition |
IRE1 | Inositol-requiring enzyme 1 |
iTRAQ | Isobaric tags for relative and absolute quantitation |
PERK | Protein kinase R (PKR)-like endoplasmic reticulum kinase |
PVDF | Polyvinylidene fluoride |
SCX | Strong cation exchange |
SDS-PAGE | Sodium dodecyl sulfate polyacrylamide gel electrophoresis |
Tm | Tunicamycin |
TOF | Time-of-flight |
UPR | Unfolded protein response |
XBP1 | X-box binding protein 1 |
Footnotes |
† Electronic supplementary information (ESI) available. See DOI: 10.1039/c7mo00114b |
‡ Equal contribution. |
This journal is © The Royal Society of Chemistry 2018 |