Platinum Binds Proteins in the Endoplasmic Reticulum of S. cerevisiae

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Platinum binds proteins in the endoplasmic reticulum of S. cerevisiae and induces ER stress Rachael M. Cunningham, and Victoria J. DeRose ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.7b00553 • Publication Date (Web): 11 Sep 2017 Downloaded from http://pubs.acs.org on September 12, 2017

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Platinum binds proteins in the endoplasmic reticulum of S. cerevisiae and induces ER stress Rachael M. Cunningham and Victoria J. DeRose Department of Chemistry and Institute of Molecular Biology, University of Oregon, Eugene, Oregon 97403, United States [email protected] Abstract Pt(II)-based anticancer drugs are widely used in the treatment of a variety of cancers, but their clinical efficacy is hindered by undesirable side effects and resistance. While much research has focused on Pt(II) drug interactions with DNA, there is increasing interest in proteins as alternative targets and contributors to cytotoxic and resistance mechanisms. Here we describe a chemical proteomic method for isolation and identification of cellular protein targets of platinum compounds using Pt(II) reagents that have been modified for participation in the 1,3 dipolar cycloaddition “click” reaction. Using this method to visualize and enrich for targets, we identified 152 proteins in Pt(II)-treated Saccharomyces cerevisiae. Of interest was the identification of multiple proteins involved in the ER stress response, which has been proposed to be an important cytoplasmic mediator of apoptosis in response to cisplatin treatment. Consistent with possible direct targeting of this pathway, the ER stress response was confirmed to be induced in Pt(II)treated yeast along with in vitro Pt(II)-inhibition of one of the identified proteins, protein disulfide isomerase.

Introduction The discovery of the antiproliferative properties of the small molecule cis-dichlorodiammineplatinum (cisplatin) in 1965 revolutionized the treatment of some cancers, most notably testicular cancer.(1) Today, cisplatin and its other FDA-approved derivatives, carboplatin and oxaliplatin, are used in over half of all cancer treatment regimens.(2) Despite the ubiquitous use of Pt(II)-based anticancer drugs, acquired resistance to the drug and side effects such as nephrotoxicity, peripheral neuropathy, and ototoxicity limit their effectiveness. Cisplatin is widely known to bind genomic DNA, activating DNA repair-mediated apoptotic pathways.(3) However, it is estimated that only 1-10% of administered cisplatin actually reaches genomic DNA,(4,5) sparking interest in investigating non-DNA targets of cisplatin. More recent research has also investigated the role of Pt(II) binding to cellular RNA, with evidence that Pt(II) readily binds to mRNA and ribosomal RNA.(6,7) In addition to nucleic acid targets, Pt(II) interactions with proteins are of great interest. It has been hypothesized that Pt(II) binding to proteins may play important roles in toxicity and resistance mechanisms.(8) Many Pt(II)-protein interactions have been characterized in vitro, using model proteins such as serum albumin, RNase A, ubiquitin, or hen egg white lysozyme.(reviewed in 8–10) Through these studies, information about the kinetics of Pt(II)-adduct formation on ACS Paragon Plus Environment

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different residues and Pt-induced changes in protein structure have been elucidated. These studies demonstrate the potential for Pt to influence protein structure and function in controlled in vitro conditions, highlighting the need for further investigation in complex cellular environments. There are few comprehensive studies of Pt(II)-protein binding in cellulo, in part due to the signal to noise challenge inherent in the low level of Pt(II) adducts. Two reports have analyzed the proteome of cisplatin-treated E. coli using multidimensional protein identification (MudPIT) following by tandem mass spectrometry to identify specific Pt adducts on peptides.(11,12) No enrichment for Pt-bound proteins was performed prior to mass spectrometry. The authors identified Pt-binding sites on proteins involved in DNA repair, ribosomal proteins, and abundant enzymes. However, as there was no enrichment prior to analysis, this method may be biased towards highly-abundant proteins and does not allow for identification of less abundant proteins that may also be platinated. Approaches to enrich for Pt-bound proteins before identification have included separation by 2-D electrophoresis or size exclusion chromatography followed by Pt detection by LA-ICP-MS (13,14) or detection of a fluorescent Pt analog (15). These have been applied to cisplatin-treated pig renal proximal tubule cells (13), rat kidney tissue.(14), and A2780 human ovarian carcinoma cells (15). These studies employ separate steps for protein separation, Pt detection, and then protein identification. While proteins were identified from the gel bands or fractions that had a Pt signal, specific Pt-bound peptides were not reported that would identify the actual platinum binding site. The authors of these studies note that this is likely due to the relatively low abundance of Pt-peptides in comparison to unmodified peptides, highlighting the further need for enrichment strategies to better identify sites of protein platination. While previous studies have provided important insight into possible Pt-protein interactions following in-cell treatment, the methods used for enrichment and isolation of specifically targeted proteins require multiple steps and specialized equipment, and overall are challenging for high-throughput studies. Here, we propose a new method of Pt-protein enrichment for MS/MS identification using azide-modified Pt reagents and the Cu-catalyzed azide-alkyne cycloaddition (CuACC) or ‘click’ reaction. This method uses readily available reagents and is easily adapted for high-throughput analysis of Pt-protein interactions in cellulo. Previously, we have described the utility of click chemistry to label Pt-bound bovine serum albumin in a simple in vitro context.(16) The goal of the present work is to apply this method in an in cellulo context to gain insight into the full spectrum of Pt-protein interactions. To accomplish this, we chose Saccharomyces cerevisiae as our model. The conservation of many important eukaryotic stress response pathways and available powerful genetic tools make yeast an excellent model for this purpose. Previous work in our laboratory has characterized levels of Pt-RNA and Pt-DNA adducts and cell death in cisplatin-treated S. cerevisiae.(6,7) To our knowledge, there are no reports exploring Pt(II)-protein interactions in yeast.



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Here we report a chemical proteomics method using click chemistry-enabled Pt(II) compounds to selectively label and isolate platinated proteins in treated yeast. Following treatment with azidoplatin (AzPt), an azide-modified Pt(II) reagent, extracted proteins can be labeled with a fluorophore for in-gel visualization, or biotin for streptavidin enrichment and analysis by LCMS/MS (Figure 1). Through this Pt(II)-pull-down method, we identified 152 proteins that are

Figure 1. (A) Structure of FDA-approved cisplatin and the two azide-modified derivatives used in this study, 2-ADAPPt and azidoplatin (AzPt). (B) Workflow for labeling of cellular proteins by click chemistry. S. cerevisiae were treated with AzPt and extracted proteins labeled with a fluorescent tag or biotin using the click reaction.

significantly enriched by pull-down from AzPt-treated samples. The isolated proteins include some previously identified protein targets of Pt(II), as well as multiple proteins that are involved in stress response pathways. Of note, 7 proteins identified to bind Pt(II) are directly involved in the ER stress response, which we confirmed was initiated in AzPt- and cisplatin-treated yeast. The ER-localized protein folding chaperone protein disulfide isomerase was identified as enriched in AzPt-treated samples, and we found Pt inhibition of protein disulfide isomerase (PDI) in vitro. Labeling Pt-bound proteins from AzPt-treated S. cerevisiae using click chemistry Previous work in our lab has characterized Pt(II) binding to RNA in treated yeast using click chemistry and modified Pt reagents. Ribosomal and transfer RNA from yeast treated with picazoplatin, an azide-derivative of picoplatin, were detected with AlexaFluor 488 DIBO.(7) Yeast treated with another azide derivative, 2-ADAPPt, also showed fluorescent labeling of rRNA and persistence of the Pt(II)-adducts on rRNA up to two hours after transfer to drug-free media.(16) These studies establish the use of click chemistry-enabled Pt(II) reagents for studying interactions of Pt(II) in S. cerevisiae. For the current studies, we chose another azide-modified derivative, AzPt (Figure 1), that is effective for in vitro and in-cell fluorescence detection post-treatment.(17) To establish labeling to Pt-bound proteins with this reagent, S. cerevisiae were treated with 75 µM AzPt for 6 hours prior to lysis. Cellular lysate was then heated in 0.15% SDS to denature proteins prior to labeling with rhodamine B-alkyne using the Cu-catalyzed click reaction. We have observed increased


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labeling by the click reaction using this method (Supplemental Figure 1), likely due to increased accessibility of Pt(II) binding sites. SDS-PAGE analysis reveals robust fluorescent labeling in AzPt-treated samples, whereas lysates from untreated yeast that underwent the click reaction do not show any labeling (Figure 2A). Many proteins from AzPt-treated samples exhibit fluorescence, indicating that AzPt has multiple targets as expected. We then performed the click reaction on lysates from AzPt-treated cells using biotin-alkyne. Following labeling with biotin, samples were incubated with streptavidin beads. Little to no protein is observed (by Coomassie stain) eluted from beads in samples from untreated yeast, but we observe multiple proteins eluting from beads incubated with samples from AzPt-treated yeast (Figure 2B). Comparing elution bands in the biotin-labeled samples with those from fluorescently-labeled samples shows that both methods label similar proteins. Additionally, although both types of labeling detect proteins that coincide with abundant proteins observed in untreated samples, the band intensities are not identical, indicating that Pt(II) targets both abundant and non-abundant proteins.

Figure 2. Fluorescent labeling and isolation of Pt(II)-bound proteins extracted from AzPttreated yeast. S. cerevisiae were treated with 0 or 75 uM AzPt for 6 hours and protein extracted and reacted with either rhodamine B-alkyne or biotin-alkyne. (a) SDS-PAGE analysis of fluorescently labeled protein shows labeling only after treatment with AzPt; no labeling is observed in untreated samples. (b) Following biotin conjugation, Pt(II)-bound proteins from AzPt treated yeast bind to and are eluted from streptavidin-coated beads. Protein that did not bind to beads after incubation was collected as “flow through” (FT). Little to no protein appears to bind to and elute from beads in untreated samples.

LC-MS/MS analysis of Pt(II)-bound proteins in AzPt-treated S. cerevisiae



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To identify Pt(II)-bound proteins extracted from AzPt-treated yeast, lysates from yeast treated with 75 uM AzPt and untreated controls were click-labeled with biotin and incubated with streptavidin-coated beads (including the denaturation step as in Figure 2B [see SI]). Following extensive washes, bound protein was then digested on-bead, and recovered peptides analyzed by LC-MS/MS. The same procedure was followed for control samples from untreated yeast. Triplicates of treated and untreated samples were analyzed. Proteins were identified against the S. cerevisiae UniProt database and validated proteins with more than 2 spectral counts were considered. Several proteins that nonspecifically bound to streptavidin beads in untreated controls were of sufficient population to be identified, which has been observed previously.(18) Approximately two times as many validated proteins were identified in AzPt-treated samples than in samples from untreated controls (Figure 3 and Supplemental Table 1). To establish statistically valid targets in AzPt-treated samples, identified proteins from both samples were normalized using normalized spectral abundance factor (NSAF)(19) and proteins from AzPt-treated samples that were significantly enriched following normalization (enrichment greater than 2-fold and P-value < 0.05) were considered as true targets. In total, we identified 152 significantly enriched proteins in AzPt-treated yeast (Figure 3A). The majority of proteins identified in pull-down samples from

Figure 3. Overview of proteins enriched by biotinstreptavidin pull-down from AzPt-treated yeast. (a) Diagram of identified proteins in treated and untreated samples. Blue circle represents 658 proteins identified in treated samples. Orange circle represents 444 proteins identified in untreated samples. Green circle represents 152 proteins from treated samples that are enriched >2-fold (P50,000 proteins/cell),whereas the significantly enriched proteins isolated from AzPt-treated samples are more evenly distributed in abundance (Figure 3B). While we identified many proteins involved in many different cellular processes, the discussion below focuses on a selection of highly enriched proteins (>3-fold enrichment) that are involved in stress responses or have been previously proposed to bind Pt(II). Confirmation of previously identified proteins that are targeted by Pt(II) Our resulting list of ‘significantly-enriched’ Pt(II)-bound proteins includes some entries that have been identified to bind platinum in previous studies (Table 1), and several novel entries. Pt(II) modification on methionine of E. coli elongation factor Tu was observed in two Table 1. Pt(II)-bound proteins from AzPt-treated cells that have been identified in previous studies. Protein

Description

DbpA

ATP-dependent RNA helicase Histidine biosynthesis bifunctional

a

a

HisI

protein

Tuf1

Elongation factor Tu 1

Tuf1

Elongation factor Tu 1

Mdh

Malate dehydrogenase

Mdh

Malate dehydrogenase

TPM2

Beta-tropomyosin

TUBA1A

Tubulin alpha-1A chain Aldehyde dehydrogenase,

a b a b

c c

c

ALDH2

mitochondrial

TrxA

Thioredoxin 1

TXN

Thioredoxin

CTSD

Cathepsin D

PABPC1

Polyadenylate-binding protein 1 Eukaryotic translation initiation factor

a

d d d

d

EIF5A

5A-1

ATOX1

Copper transport protein Transitional endoplasmic reticulum

VCP

e

PDIA4

e

d

ATPase

f

Protein disulfide isomerase A4

f f

PDIA4

Protein disulfide-isomerase A4-like

PDI-P5

Protein disulfide isomerase P5

PDIA3

Protein disulfide-isomerase A3

g

P4HB

PDIA6 PDIA3

g g

Protein disulfide isomerase A1 Protein disulfide isomerase A6 Protein disulfide isomerase A3

c d

h h h

Accession

Organism

Protein identified in this study

P21693

E. coli

DBP2 + DED1

P06989

E. coli

HIS4

A7ZSL4

E. coli

TUF1 + TEF1

A7ZSL4

E. coli

TUF1 + TEF1

P61889

E. coli

MDH3

P61889

E. coli

MDH3

A1X899

Renal proximal tubule epithelial cells (Sus scrofa)

TPM1

P02550


TUB2

Q2XQV4


ALD4 +ALD5

P0AA25

E. coli

TRX1 + TRX2

P11232

Kidney tissue (Rattus norvegicus)

TRX1 + TRX2

P24268


PEP4

Q9EPH8


PAB1

Q3T1J1


HYP2

Q9WUC4


ATX1

P55072

HEI-OC1 cell line (Mus musculus)

CDC48

P13667

HEI-OC1 cell line (Mus musculus)

PDI1

F1SAD9


PDI1

E1CAJ6


PDI1

P11598


PDI1

P07237

A2780 cell line (Homo sapiens)

PDI1

Q15084


PDI1

P30101


PDI1

Reported are closest matched yeast homologs to originally identified protein. Highlighted in green, yellow, blue, and purple are proteins that have been predicted bind Pt(II) in more than one previous study. Also included are identified Pt(II)binding sites on specific residues. Studies were performed using cisplatin unless marked otherwise (e CFDA-Pt; g 2NH3Pt-agarose conjugate). See reference for details (a 11; b 12; c 13; d 14; f 21; h 20).



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separate studies,(10,11) and we observe both mitochondrial elongation factor tu (TUF1) and eukaryotic elongation factor 1a (eEF1a) enriched in pull-downs from our AzPt-treated samples. Malate dehydrogenase, which is enriched in our treated samples, is also modified in cisplatintreated E. coli at threonine and serine residues.(10,11) Both thioredoxins (TRX1 and TRX2) are enriched in treated samples (Table 2, yellow), and were also proposed to be platinated in kidney tissue from cisplatin treated rats,(13) and modified at His7 in E. coli thioredoxin.(26) Various isoforms of protein disulfide isomerase (PDI) have been identified in multiple cell lines following treatment with Pt(II)-based compounds.(12,13,21,15) Yeast PDI1 was significantly enriched in AzPt-treated samples, providing further evidence that PDI is targeted by Pt(II). We observed pull-down of selected metal-binding proteins, most notably copper ion chaperones and transporters (Table 2, green). Of note, we identified copper chaperones ATX1 and FET3 as being significantly enriched in AzPt-treated samples. ATX1 is responsible for the trafficking of Cu(I) from copper transporter 1 (CTR1) to the multicopper oxidase FET3 in the golgi apparatus.(22) ATX1 contains the metal-binding domain CXXC, which has been shown to bind Pt(II) following cisplatin treatment in vitro.(23–26) One way in which cisplatin and other Pt(II)based drugs enter the cell is by active transport through CTR1.(27) The identification of these copper binding proteins in this Pt-pull-down approach adds to the evidence that this is one mechanism through which Pt(II) influx and efflux is mediated. ER stress-related proteins are targeted by Pt(II) Also of interest was the identification of multiple proteins involved in endoplasmic reticulum (ER) stress. The ER is the main site of post-translational modifications and folding of secretory proteins, and perturbations to its function result in an accumulation of unfolded proteins in the lumen of the ER. This elicits the unfolded protein response (UPR), an adaptive response which ultimately results in the increased expression of protein folding chaperones, reduction in global translation, and increased degradation of accumulated unfolded proteins.(28) The ER stress Table 2. Subset of proteins that are involved in stress responses and are enriched by Pt(II) pull-down from AzPt-treated S. cerevisiae. Protein

Description

Enrich P-Value

CDC48

Cell division control protein 48

∞

1.02E-05

PDI1 PRE9 PUP2

Protein disulfide isomerase Proteasome subunit alpha type-3 Proteasome subunit alpha type-5

6.2 3.8

7.90E-03 8.21E-04 2.74E-02

Response to stress/ ER associated misfolded protein catabolic process Response to stress/Protein folding Proteasomal protein catabolic process Proteasomal protein catabolic process

RPN10 26S proteasome regulatory subunit RPN10

∞

5.55E-03

Proteasomal protein catabolic process

RPN2 RSP5 ATX1

26S proteasome regulatory subunit RPN2 E3 ubiquitin-protein ligase RSP5 Metal homeostasis factor ATX1

∞ ∞ ∞

3.40E-04 6.17E-03 1.01E-04

Proteasomal protein catabolic process Proteasomal protein catabolic process Response to stress/Copper chaperone activity

CCS1 Superoxide dismutase 1 copper chaperone

∞

GO biological process

4.6

3.18E-02

Response to stress/Intracellular copper ion transport

CUP1-1 TRX1 TRX2

Copper metallothionein 1-1 Thioredoxin-1 Thioredoxin-2

6.3 2.1 3.7

4.48E-03 2.36E-02 2.71E-02

NHP6A

Non-histone chromosomal protein 6A

∞

4.96E-03

Response to stress/Detoxification of copper ion Response to stress/Cell redox homeostasis Response to stress/Cell redox homeostasis Response to stress/DNA replication-independent nucleosome organization

POL30

Proliferating cell nuclear antigen

∞

3.84E-03

Response to stress/Nucleotide excision repair

Enrichment value is reported as fold enrichment in AzPt treated samples following normalization in all samples (∞, protein was not identified in control samples). Listed proteins are grouped by response: pink=UPR and ER-associated degradation green=metal homeostasis yellow=cell redox homeostasis blue=DNA damage response


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response is initially pro-survival, but can be pro-apoptotic if homeostasis is not restored.(29) Multiple proteins belonging to the 26S proteasome, the E3 ubiquitin ligase RSP5, protein disulfide isomerase (PDI), and cell division control protein 48 (CDC48) were pulled down in AzPt-treated samples (Table 2, pink). One of the most significant identified proteins was CDC48, which had high spectral counts in all treated samples and was not identified in untreated samples. High protein levels of the human homolog of CDC48, VCP/p97, have been observed in cancerous cells and it is proposed to play a role in tumorigenesis.(30) Modification of Cys522 in VCP by a small molecule has been shown to inhibit its ATPase activity, resulting in cell death.(31) As Pt(II) is also reactive towards cysteines, it would be interesting to investigate whether Pt(II) binds to Cys522 and affects VCP function. The consistent identification of PDI across multiple studies, including our own, suggests that PDI may be a significant target of Pt(II) in multiple cell types. PDI is an important protein folding chaperone found in abundance in the lumen of the ER, where it plays an essential role in ensuring proper folding of secretory proteins by catalyzing the formation and rearrangement of disulfide bonds. Disruption in PDI function result in the accumulation of unfolded proteins, and ER stress. Additionally, when the cell enters ER stress, PDI expression is increased in an attempt to restore proper folding of proteins in the ER. Increased PDI expression and activity are observed in many cancers, indicating that cancer cells may be more sensitive to PDI inhibition.(32) Because of this, the development of therapeutics that target PDI have been of particular interest.(33–36) The active site of PDI contains a thioredoxin-like domain, CXXC, and modifying these cysteines using small molecules results in inhibition and cell death.(33,36,37) Recently, PDI has been proposed to play a role in cisplatin resistance, as knockdown of different isoforms of PDI in both lung adenocarcinoma and ovarian carcinoma cells sensitizes resistant cells to cisplatin.(38,39) We hypothesize that Pt(II) may bind to cysteines in the active site of PDI, thereby inhibiting its function and ultimately resulting in the induction of ER stress. It has been well established that cisplatin and other Pt(II)-based drugs induce ER stress in mammalian systems,(40) however the mechanisms and responses remain unclear. It is possible that Pt(II) adduct formation on proteins important for the maintenance of proteostasis could inhibit their functions by causing structural alterations or directly binding to active sites in the protein. Pt(II) binds to and inhibits PDI in vitro To answer the question of how Pt(II) binding effects PDI function, we started by investigating this in an in vitro system. PDI was incubated with 1 or 2 molar equivalents of cisplatin or the azide-modified 2-ADAPPt, and then assayed for reductase activity. 2-ADAPPt has been employed in previous studies investigating Pt-protein, Pt-DNA, and Pt-RNA binding. 2-ADAPPt and AzPt differ only in the addition of the peptide linkage in AzPt, and it has been shown that both compounds have similar binding yields to a DNA hairpin oligo, but that AzPt has higher labeling yields when used in the click reaction.(17) As we are interested mainly in the effects of Pt binding to protein, we chose to use 2-ADAPPt for these in vitro experiments. We observe dose-dependent inhibition of PDI treated with both cisplatin and 2-ADAPPt (Figure 4A). Cisplatin appears to be a potent inhibitor, as PDI treated with 2 molar equivalents cisplatin has 51% activity of untreated PDI after 45 min. We observe lesser inhibition of PDI treated with 2 molar equivalents of 2ADAPPt, with 73% activity of the untreated control at 45 min.



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While both Pt(II) compounds inhibit PDI, the difference in inhibition between cisplatin and 2ADAPPt indicates that cisplatin may bind to PDI more readily than 2-ADAPPt. We confirmed the presence of Pt(II) on PDI following treatment with 2-ADAPPt using the click reaction to attach alkyne-rhodamine B (Figure 4B). The ratio of fluorescence intensity to Coomassie staining for 1 eq. (0.41) vs. 2 eq. (0.98) Pt reveals an approximate 2.4-fold increase in fluorescence of 2 eq. 2ADAPPt-treated PDI (Supplemental Table 3). Dose-dependent fluorescent labeling of 2-ADAPPttreated PDI is observed, indicating that 2-ADAPPt does indeed bind to PDI in vitro.

Figure 4. Pt(II) reagents bind to and inhibit PDI. (A) PDI treated with 1 and 2 equivalents of cisplatin and 2-ADAPPt shows inhibition of activity over time. PDI activity was measured by PDI-catalyzed reduction of insulin in the presence of DTT. Reactions were stopped at 5, 25, and 45 minutes and insulin aggregation measured. Values graphed as average (n=3) ± SD. (B) Fluorescent labeling of 2-ADAPPttreated PDI using the click reaction. PDI was treated with 0, 1, or 2 molar equivalents of 2-ADAPPt. Pt(II)-bound PDI was then reacted with Rho-B alkyne, and resolved by SDS-PAGE, followed by fluorescent imaging and coomassie staining to detect protein. Increased fluorescence is observed with increasing molar equivalents of 2-ADAPPt.

Induction of ER Stress in AzPt-treated S. cerevisiae While ER stress has been shown to be induced following cisplatin treatment in mammalian cells, this response has not been investigated in S. cerevisiae. We were therefore interested in determining whether ER stress is induced in S. cerevisiae during treatment with cisplatin and AzPt. ER stress and the UPR are sensed through only one protein in yeast, IRE1.(41) IRE1 is a transmembrane protein localized in the ER that upon accumulation of unfolded proteins, activates an endoribonuclease domain which is responsible for the unconventional splicing of HAC1 mRNA.(42) HAC1 protein then acts as a transcription factor that induces expression of chaperones and proteasomal subunits to help clear the ER of unfolded proteins.(43) As this is a very specific


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response during the yeast UPR, HAC1 mRNA splicing can be monitored to determine if the UPR has been activated.(44) Yeast were treated for 6 hours with either 75 uM AzPt, 200 uM cisplatin, or 2 mM dithiothreitol (DTT), which is a known inducer of the UPR. HAC1 splicing status was then determined by RT-PCR (Figure 5A). We observe very little HAC1 splicing in untreated samples and very robust splicing of 48% in DTT treated samples. Yeast that were treated with 200 uM cisplatin show 38% splicing of HAC1. In comparison, 31% splicing was observed in 75 M AzPttreated yeast. These results show that HAC1 splicing and the UPR are indeed induced in yeast treated with either AzPt or cisplatin. ER stress and the UPR is initially a pro-survival response, and only once the stress is prolonged or severe enough does it become apoptotic.(41) We next determined the cell viability of AzPt-treated yeast by clonogenic assay (Figure 5B). Yeast treated with 75 uM AzPt for 6 hours show very little reduction in cell viability, remaining with 83% viability following treatment. We compared this to cisplatin treated yeast, which show viability of 53% and 27% after 100 and 200 uM treatment, respectively; these values agree with previous reports.(6) These results indicate that

Figure 5. 75 µM AzPt induces ER stress but not cell death in yeast. (a) RT-PCR of HAC1 mRNA on total RNA extracted from yeast treated with 200 µM cisplatin, 75 µM AzPt, 2 mM DTT, or left untreated for 6 hours. Higher molecular weight band is the uninduced mRNA (HAC1u), lower molecular weight band appears under ER stress as the spliced, or induced form (HAC1i). Values of percent HAC1 mRNA spliced reported as the average ± SD (n=3). (b) Clonogenic assay of yeast treated with 75 µM AzPt and 100 or 200 µM cisplatin for 6 hours. Percent colony forming units were calculated by comparing to untreated controls and values reported are the average ± SD (n=3).

while AzPt can induce the UPR, as indicated by HAC1 splicing, this response is likely still in the pro-survival phase of the response at the assayed treatment conditions. Because of this, we are likely capturing the early response, which would be to our benefit as we wish to understand the initial events that induce the UPR during treatment.



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Conclusions Despite decades of reports of Pt(II) binding to proteins, very little is understood about these interactions in cells and how they contribute to the mechanism of action of Pt(II) drugs. This lack of understanding is exacerbated by the challenge of identifying Pt(II)-protein interactions in cellulo. We have applied a chemical proteomics approach using click-enabled Pt(II) compounds to investigate these interactions. We have used this method to enrich for Pt(II)-bound proteins in S. cerevisiae treated with AzPt, simplifying downstream analysis by mass spectrometry. The identification of some previously predicted Pt(II)-binding proteins indicates that AzPt likely targets similar proteins as cisplatin and validates our method. We also identified some new and potentially important targets of Pt(II), including proteins involved in ER stress. It is important to note that cisplatin treatment has been shown to result in changes to the proteome in mammalian cancer cell lines. Therefore, it is possible that increased pull-down of some proteins in AzPt-treated samples in comparison with untreated samples may be due to increased expression of those proteins under Pt(II) treatment and higher nonspecific interaction with streptavidin beads. Previous proteomics studies in nasopharyngeal(45) and ovarian(46) carcinoma cell lines, as well as cochlear(47,48) and renal(49) cells, have shown that for the most part, cytoskeletal proteins (actin, tubulin, vimentin), proapoptotic proteins (caspase 3), and heat shock proteins (HSP70, HSP60) are increased with cisplatin treatment. One study in nasopharyngeal carcinoma cells found an upregulation of proteasome subunit alpha type 3 following cisplatin treatment, indicating that this identification in our data should be further validated. Interestingly, however, PDI proteins are found to be downregulated (up to 5-fold),(45,49) or have no significant change in expression.(47) VCP (CDC48) was also found to be downregulated in cisplatin-treated cochlear cells.(47) This increases confidence in identification of ER stress-related CDC48 and PDI in our pull-down studies as true targets of Pt(II). Current work is focused on implementing this methodology in mammalian cancer cell lines to investigate differences in protein targeting by Pt(II) in different cell lines. Using an in vitro assay, we confirmed binding of Pt(II) to PDI and found significant inhibition of PDI reductase activity following Pt(II) treatment. Additionally, we observe induction of the UPR in Pt(II)-treated yeast as indicated by HAC1 splicing. Induction of ER stress in mammalian cells during Pt(II) treatment has been proposed to play an important role in cytotoxicity,(40) however the mechanism through which Pt(II) induces the UPR is still unknown. From the data presented here, we hypothesize that Pt(II) binding to PDI and subsequent inhibition contributes to induction of the UPR during treatment with platinum compounds. Methods Cell Cultures and Pt(II) Treatment. S. cerevisiae strain BY4741 (MATa; his3Δ1; leu2Δ0; met15Δ0; ura3Δ0) was grown in synthetic complete (SC) media (0.67% yeast nitrogen base, 2% glucose) supplemented with amino acids at 30°C with shaking. OD600 of overnight cultures were determined and used to inoculate AzPt or cisplatin containing media to an OD600 of 0.1. AzPt was synthesized as described previously.(17) Due to poor solubility in aqueous media, AzPt-containing media was prepared by adding AzPt to 50 mL SD broth and shaking at 30°C overnight. Final concentration of AzPt in media was determined by ICP-MS following dilution in 3% HNO3. For cisplatin treatments, 5 mM cisplatin was prepared fresh in milli-Q water and added at indicated concentrations to SD broth immediately before inoculation. Following inoculation, yeast were


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allowed to grow at 30°C with shaking for 6 hours. Cells were then collected by spinning down at 4000 RPM for 10 min at 4°C and cell pellets stored at -30°C until lysis. Cell Viability Experiments. Yeast were inoculated to an OD600 of 0.1 in SD broth containing either 75 µM AzPt, 100 µM cisplatin, or 200 µM cisplatin. After 6 hours, OD600 measurements were used to calculate cell growth (1 AU=2x107 cells/mL). Cell viability was determined by plating serial dilutions of treated and untreated yeast on YEPD plates (2% peptone, 1% yeast extract, 2% glucose, 2% agar). Colonies were counted following growth for 3 days at 30°C. Colony forming units were calculated by dividing the number of colonies in treated samples by the number of colonies in untreated samples. 3 biological and 3 technical replicates were used for each condition. HAC1 mRNA Splicing Analysis of Treated Yeast. Yeast were treated and pelleted as described previously. Concentrations of 100 and 200 µM cisplatin, 75 µM AzPt, and 2 mM dithiothreitol (DTT) were used for treatments. RNA was extracted from yeast pellets using MasterPure yeast RNA purification kits (Epicentre, MC85200) and treated with Turbo DNase (Thermo Fisher, AM1907) according to manufacturer’s protocols. DNase reactions were cleaned up by ethanol precipitation and RNA resuspended in milli-Q water. For reverse transcription of poly adenylated mRNA, 500 nmol of RNA was reverse transcribed by SuperScript II (Thermo Fisher, 18064) using oligo dT primer. Resulting cDNA was then amplified using HAC1 forward (5’- CTG TAC AAT GGA GCC TGC GA -3’) and reverse (5’- AAA TGA ATT CAA ACC TGA CTG CG -3’) primers. PCR products were separated on 2% agarose gel and visualized by ethidium bromide staining. For quantification of splicing, GelAnalyzer (www.gelanalyzer.com) was used for densitometry of spliced and unspliced bands. Percent splicing was calculated by diving the intensity of the spliced (HAC1i) band over the total intensity of both bands (HAC1u + HAC1i). 3 biological replicates were used for each treatment condition. Click Chemistry on Yeast Lysate. Lysates were prepared by adding acid-washed glass beads (0.2 µM, Sigma-Alrich, G8772) and “breaking buffer” (50 mM sodium phosphate buffer (pH 7.4), 5% glycerol) with fungal protease inhibitor cocktail (Sigma Aldrich, P8215) to the cell pellets. Cells were then mechanically lysed by vortexing 30 seconds and placing on ice for 30 seconds, repeating a total of 5 times. Debris was spun down at 14000 xg for 10 minutes. Supernatant was collected and protein concentration was determined using Bradford reagent. Lysates were kept at -30°C until use. For click chemistry, 70 µg of lysate was first denatured by adding 0.15% SDS and heating at 90°C for 2 minutes. Using this denaturing step prior to the click reaction, we have observed significant increases in labeling yield (supplemental figure 1), likely due to exposure of more buried platination sites. Rhodamine B-alkyne was synthesized as described previously.(17) Denatured lysate was reacted with either 66 µM rhodamine B-alkyne or biotin-alkyne (Sigma Aldrich, 764213) with 1.2 mM CuSO4, 12 mM THPTA, and 1.2 mM sodium ascorbate for 1 hour at room temperature. Reactions were then cleaned up using laboratory prepared spin columns packed with Sephadex G-25 medium (Sigma Aldrich). For rhodamine-B labeled lysate, samples were resolved by 10% SDS-PAGE, followed by imaging of fluorescent labeling using an AlphaImager system, and staining by coomassie brilliant blue. Biotin-labeled lysates were then added to 60 µL of agarose streptavidin beads (Thermo Fisher, 20357). Beads were incubated overnight at 4°C with rotation. Unbound protein was collected, beads were washed five times with PBS, and proteins were eluted in 2% SDS loading buffer. Prior to loading on the gel, unbound fraction (“flow through”) and input (biotin labeled lysate that had not been incubated with beads)



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were reduced in volume to 30 µL by SpeedVac. Half of “flow through” and “input” fractions and entirety of elution fraction were resolved by 10% SDS-PAGE. Protein was stained using coomassie brilliant blue. Preparation of Biotin Labeled Yeast Lysate for LC-MS/MS. Triplicate biological replicates of 250 µg of AzPt-treated or untreated yeast lysate was reacted with biotin-alkyne for labeling as described above. Reactions were reduced to 300 µL by SpeedVac and incubated with 200 µL agarose streptavidin beads overnight at 4°C with rotation. Unbound protein was collected, and beads were washed three times with PBS. Beads were then reconstituted in 200 µL 50 mM ammonium bicarbonate (Sigma Aldrich, A6141) and transferred to a fresh tube. Three additional washes in 50 mM ammonium bicarbonate were performed. Following the third wash, bound proteins were reduced in 5 mM DTT in ammonium bicarbonate followed by carbamidomethylation using 114 mM iodoacetamide (Sigma Aldrich, I6125). Digestion was performed using 2 ug porcine trypsin (Sigma Aldrich, T6567) overnight. Digestions were acidified to 2% formic acid, and removed from beads. Beads were washed once more with 50 mM ammonium bicarbonate and this fraction was added to final digest and volume was reduced to 100 µL by SpeedVac. Digestions were cleaned up by adding to C18 spin columns (Thermo Fisher, 89873) washed with 0.5% trifluoroacetic acid and 5% acetonitrile, followed by elution in 40 µL 70% acetonitrile. Eluents were dried to completion by SpeedVac and submitted to the UC Davis Proteomics Core for LC-MS/MS analysis. LC-MS/MS Protein Identification. LC-MS/MS data were received as raw files which were converted using MS Convert.(53) Database searching was performed using X!Tandem and MyriMatch through Search GUI.(54) Uniprot database for S. cerevisiae was used and amended with reversed sequences for calculation of false discovery rate. Carbamidomethylation of cysteine was set as a fixed modification and oxidation of methionine was set as a variable modification. MS precursor tolerance was set to 10 ppm and fragment tolerance was set to 0.5 Da. Proteins were validated by Peptide Shaker.(55) Only proteins with >99% confidence and >2 spectral matches were considered. Normalized spectral abundance factor (NSAF) of validated proteins were calculated and used for further analysis. Proteins with >2 fold enrichment and P-value

Platinum Binds Proteins in the Endoplasmic Reticulum of S. cerevisiae

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