TRAP1 binds to glutamine synthetase and might affect cancer cell metabolism.

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TRAP1 binds to glutamine synthetase and might affect cancer cell metabolism.

Lim, Hakmyeong

Department of Biological Sciences Graduate school of UNIST

2015

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TRAP1 binds to glutamine synthetase and might affect cancer cell metabolism.

Lim, Hakmyeong

Department of Biological Sciences

Graduate school of UNIST

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TRAP1 binds to glutamine synthetase and might affect cancer cell metabolism.

A thesis

Submitted to the Graduate School of UNIST in Partial fulfillment of the requirements for

the degree of Master of Science

Lim, Hakmyeong

12. 19. 2014.

Approved by

_______________________________

Major advisor

Byoung Heon Kang

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TRAP1 binds to glutamine synthetase and might affect cancer cell metabolism.

Lim, Hakmyeong

This certifies that the thesis of Lim, Hakmyeong is approved.

12. 15. 2014.

_________________________________

Thesis supervisor: Byoung Heon Kang

_________________________________

Byoung Heon Kang

_________________________________

Chan Young Park

_________________________________

Tae Joo Park

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Abstract

TRAP1 is anti-apoptotic mitochondrial protein. There have been implications about this chaperone and cancer cell metabolism but it is not known how TRAP1 affects. Glutamine synthetase is a candidate of TRAP1 binding protein in this study. GS is well known as ammonia detoxifying function. This paper will deal with 2 proteins’ binding and their role in cancer cell metabolism.

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Contents

Abstract………....5

Contents………...6

List of figures and Nomenclature……….7

I-1. Introduction……….………..8

I-2. Materials and Methods……….………...9

I-2-1. GST&GST-Recombinant protein expression………...9

I-2-2. GST-pull down assay………...9

I-2-3. Cloning………...9

I-2-4. Binding assay with in vitro translation……….………...9

I-2-5. Mitochondria import assay………...10

I-3. Result………...11

I-4. Discussion………...13

Reference...15

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List of figures

Figure 1. GST-pull down assay of

TRAP1...11

Table 1. Highest ΣCoverage valued part of Mass spectrometry result...11

Figure 2. Mouse GS sequence covered by identified peptides from mass analysis...11

Figure 3. Binding assay with in vitro

translation...12

Figure 4. Mitochondrial import

assay...12

Figure 5. Similarity in amino acid sequence of Human and chicken...13

Figure 6. 2 points of view of TRAP1 and GS binding in

mitochondria...14

Nomenclature

ATP: Adenosine Triphosphate GST: Glutathione S-transferase

TRAP1: Tumor necrosis factor-Receptor Associated Protein 1 GS: Glutamine Synthetase

HEPES: 4-(2-HydroxylEthyl)-1-PiperazineEthaneSulfonic acid

SDS-PAGE: Sodium-DodecylSulfate – PolyacrylAmide Gel Electrophoresis PCR: Polymerase Chain Reaction

GLT-1: Glial glutamate Transporter-1

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CHAPTER I

The binding of TRAP1 and GS

I-1. Introduction

Cancer cell have been under study for a long time. The directions of cancer study are various such as cell cycle, apoptosis, and etc discussed by many scientists like Vermeulen (2003) and Shivapurkar (2003). One more special point about cancer cell is its metabolism. Cancer cell has different respiration mechanism with normal cell. The oxidative phosphorylation after glycolysis is most efficient and important energy source in normal cell while it is depressed in cancer cell. Glutamine metabolism compensates the ATP production. This is often called ‘Warburg effect’ by Vander Heiden (2009). It is caused by several factors. For instance, Building blocks made by glycolysis are essential for high proliferating cancer cells. Surroundings of cancer cell has not enough oxygen due to massive growth. Function of cancer mitochondria is usually inhibited by cancer genes because mitochondria is center of apoptosis as Vander Heiden (2009). Closely related to cancer cell’s characteristic and identity, shifted cancer cell metabolism can be a key to understand cancer.

One of the cancer specific protein is TRAP1 (Tumor necrosis factor-Receptor Associated Protein 1) which has undeniable role in cancer cell’s life. It is known to be Heat shock protein 90’s mitochondrial homolog, so TRAP1 can act as chaperone according to Felts (2000). It is also known that TRAP1 binds to cyclophilin D for inhibiting permeability transition pore on inner membrane, which brings out antiapoptotic situation reported by Kang (2007). One more thing is that this protein is involved in cancer cell metabolism. A recent paper said that TRAP1 deficient cell had increased mitochondrial respiration and decreased glycolysis just as report of Kang (2007). I tried to find the metabolic partner of TRAP1 in mitochondria, whose result was Glutamine synthetase (GS).

Glutamine synthetase, as its name indicates, makes glutamine from glutamate and ammonia with ATP consumption. This reaction is usually applied as nitrogen assimilation or ammonia detoxification according to Meister (1988). In liver cell, especially, this reaction occurs more than other organs, because liver is center of ammonia detoxification just as report of Lobley (1995). According to Cadoret (2002), One more interesting thing is that GS is overexpressed in HCC because of β-catenin.

In this paper, it is talked that GS can be located in cancer cell mitochondria and have possible roles in interaction with TRAP1, which might connect GS to cancer cell metabolism.

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I-2. Materials and Methods.

I-2-1. GST&GST-Recombinant protein expression

“Vacant or gene cloned pGEX-4T1” transformed BL21 E.coli was cultured with LB 500mL at 37℃

and 180 rpm horizontal shaking. When it comes to 0.4∼0.6 of OD600 value, IPTG (isopropyl-β-D- thogalactopyranoside) was put in to become 0.2mM for inducting GST or GST-fused protein. After 18℃ overnight induction, E.coli was suspended by 4℃ 3500rpm 15min centrifuge. Resided pellet was broken by sonication and cell debris was removed with 4℃ 15000rpm 30min centrifuge. The supernatant of centrifuge was fused with Glutathione-sepharose bead for binding.

I-2-2. GST-pull down assay

GST-bead and GST-TRAP1-bead were settled in open-column. Mitochondrial fraction of mouse brain and liver were obtained from 5 weeks old Balb/C strain mouse. All the reactions were done at 4℃ for protein stability. Mitochondrial proteins were added to beads with gravity flow. Protein bound beads were washed with 20mM HEPES, 2.5mM MgCl2, 75mM KCl, 1M NaCl. Washed beads were then analyzed with SDS-PAGE. Suspected protein bands come from silver staining were extracted and had In-gel digestion in order to get mass-spectrometry.

I-2-3. Cloning

Total RNA was obtained from HepG2 by QIAGEN kit. In order to increase total amount of mRNA, mRNA purification was left out. Primers surrounding GS gene with EcoRI (5’) and NotI (3’) restriction enzyme site were accompanied for Reverse transcription PCR. Other PCRs of vector pGEX-4T1 (for protein expression) and pcDNA3 (for in vitro translation) at corresponding restriction enzyme sites were also undertaken. Cleaving with each restriction enzyme were done at 37℃ for 3 hours. Purely cleaved GS gene & vector fragments were purified by agarose-gel extraction. Two fragments were ligated at 16℃ for 14hours. Transformation and Mini-prep by GeneAll kit was done for confirm whether right plasmids were cloned. Sequencing was done by Macrogen.

I-2-4. Binding assay with in vitro translation

GST and GST-TRAP1 beads were blocked with 10mg/ml BSA. 2μl of GS cloned pcDNA3 was incubated with 2μl of ³⁵S tagged methionine and 22μl of TNT T7 Quick master mix from Promega at 30℃ for an hour. 10μl of blocked beads were incubated with in vitro 10μl of translated GS, 20μl of 10mg/ml BSA, and 160μl of HEPES buffer (HEPES 20mM, KCl 75mM, EDTA 0.1mM, NP-40

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0.05%, and MgCl2 2.5mM) for overnight. Beads were mildly washed with HEPES buffer for 3 times and analyzed with SDS-PAGE. After gel running and Coomassie staining are over, gel was dried in center of cellophane film in order to stay plain. Dried gel was analyzed for radioactive signals from

35S by Typhoon 7200.

I-2-5. Mitochondria import assay

Overall procedure was followed by method of Young (2007). Another ³⁵S tagged protein expressed just as in vitro translation while 5 weeks old Balb/C mouse’s liver mitochondria is obtained.

Expressed protein is incubated at 30℃ for 20 min with mitochondria suspended in MS buffer (250 mM sucrose, 80 mM Potassium acetate, 20 mM HEPES-KOH [pH 7.5], and 5 mM Magnesium chloride). Half of the incubated solution is treated with proteinase K to become 50μg/ml at 4℃ for 1 min in order to remove unimported proteins. After washing the mitochondria with MS buffer once, SDS-PAGE was done and gel was analyzed for radioactive signals from ³⁵S by Typhoon 7200.

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I-3. Results.

←∼42kDa

←∼38kDa

←∼26kDa

Fig 1. GST-pull down assay of TRAP1. GST-bead + Liver, GST-TRAP1 bead, GST-TRAP1 bead + brain mitochondria, and GST-TRAP1 bead + liver mitochondria from left to right

Description ΣCoverage

Glutamine synthetase

OS=Mus musculus GN=Glul PE=1 SV=6 - [GLNA_MOUSE] 21.98 Actin-related protein 2

OS=Mus musculus GN=Actr2 PE=1 SV=1 - [ARP2_MOUSE] 19.29 Septin-5

OS=Mus musculus GN=Sept5 PE=1 SV=2 - [SEPT5_MOUSE] 19.24 Serpin B6

OS=Mus musculus GN=Serpinb6 PE=2 SV=1 - [SPB6_MOUSE] 11.64 Ganglioside-induced differentiation-associated protein 1-like 1

OS=Mus musculus GN=Gdap1l1 PE=2 SV=1 - [Q3USC7_MOUSE] 10.08 Mitogen-activated protein kinase 1

OS=Mus musculus GN=Mapk1 PE=1 SV=3 - [MK01_MOUSE] 9.78

Table 1. Highest ΣCoverage valued part of Mass spectrometry result. ΣCoverage is an estimated value summarized of

‘amount of covered sequence by found-peptides’, ‘confidency in peptide matching”, and ‘no existence in control band’. Glutamine synthetase was selected as a significant result from overall suspect proteins.

Figure 2. Mouse GS sequence covered by identified peptides from mass analysis

Fig 1 shows 3 bands in liver mitochondria column which was not shown in brain. After the mass spectrometry, GS, at the ~42kDa band, was the most significant candidate because of relativity in

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metabolism and cancer. The coverage of GS is shown in Fig 2.

A. B

C

Fig 3. Binding assay with in vitro translation. A. Coomassie staining image. B. Detection of ³⁵S-Methionine Each column means (³⁵S-Methionine GS)-(vacant)-(GS + GST-bead)-(GS + GST-TRAP1 bead) from left to right. C is detection of ³⁵S radioactivity of (³⁵S-Methionine TRAP1) - (TRAP1 + GST-bead) - (TRAP1 + GST-GS bead)

A. 1 2 3 B. 1 2 3 C. 1 2 3 D. 1 2 3

Fig 4. Mitochondrial import assay. A. OTC(Tom 20), B. PiC(Tom70), C. GS, and D. GS with cytosolic fraction. 1 is protein itself, 2 is protein with mitochondria, and 3 is PK treated sample of protein with mitochondria.

In vitro translation in Fig 3 showed that GS directly binds to GST-TRAP1 much more than GST while actual amount of GST-TRAP1 bead is rather less than that of GST bead. This confirms the actual binding of GS and TRAP1 in vitro. But binding of TRAP1 with GST-GS was not seen. This difference will be discussed later.

Mitochondrial import assay in Fig 4 did not show the import of GS and positive control of Tom20,

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OTC (Ornithine tramscarbamoylase), according to Takanori (2001), into mouse normal liver mitochondria while positive control of Tom70, PiC (Phosphate carrier protein), reported by Young (2007), exhibited typical MTS-cleaving import. The additional experiment incubating cytosolic fraction together was also done but, as you can see in Fig 3, there was no difference in result.

I-4. Discussion

We can see that TRAP1 directly binds to GS in vitro. It is interesting that GS binds to GST-TRAP1 while TRAP1 doesn’t bind to GST-GS. We can make 2 assumptions. One is that N-terminal sequence of GS is necessary for binding. TRAP1 in this case would act as a mitochondrial guidance or setting localization as N-terminal sequence of GS can be used as targeting sequence. This hypothesis can be supported by fact that cytosolic HSP70 and 90 guides protein to mitochondria. Another assumption is based on GS’ homopolymer. GS is known for polymerized in vivo at active state. So it can be assumed that TRAP1 only binds to polymerized form of GS. This case can deduce that binding between TRAP1 and GS is related to GS activity. These hypotheses can be solved by in vivo binding, Knock-out study and activity assay.

The import of GS into normal liver cell mitochondria could not be seen. The murine mitochondria might not import human OTC and GS. As weak mitochondrial targeting sequence of GS is at N- terminal, GS seems to be imported through Tom20 according to Brix (1999). Human cell mitochondria should be checked through this import assay.

B.

A.

Fig 5. Similarity in amino acid sequence of Human and chicken. A is whole amino acid sequence. B is N-terminal region

There are some indications that GS might get into cancer mitochondria. As reported by Matthews (2009), uricotelic species’ liver has mitochondrial GS due to their higher mitochondrial membrane potential. It is known that cancer cell’s mitochondrial membrane potential is higher than that of

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normal cell’s because of lacking oxidative phosphorylation. Moreover, uricotelic GS’ N-terminal has

“weak” mitochondrial targeting sequence. The 23 amino acids at N-terminal for human and chicken has 73.9% similarity. The most important difference is that 19^th amino acid is positive in chicken’s while not in human’s. As positive charge is important for mitochondrial import, this might be significant change.

A. B.

Fig 6. 2 points of view of TRAP1 and GS binding in mitochondria. A. is GS involved in Ammonia detoxification. B. is about glutamine metabolism related to energy production.

There is another significant thing in GS and HCC. C3A and HepG2, both of them are HCC, have little or no expression of urea cycle enzyme reported by Mavri‐Damelin (2008). As a result, those cells could not detoxify ammonia while normal hepatocytes could. As GS and GLT-1, glutamate uptake protein, according to Cadoret (2002), is overexpressed in HCC, glutamine production could be alternative ammonia detoxification with harvested glutamate from outside of cell and highly expressed GS, even though ATP is consumed. This newly made glutamine can be nitrogen source for protein, nucleotides, and etc discovered by Rajagopalan and DeBerardinis (2011). In this situation, GS has another reason for getting into mitochondria because ammonia is usually made in mitochondria.

So GS had better get into mitochondria to detoxify efficiently. TRAP1, the antiapoptotic protein of cancer cell, would help GS’ activity somehow if import of GS is for ammonia detoxification.

But there is another possibility. Glutamine metabolism initiated by making glutamate from glutamine would be bothered if GS go into mitochondria. Even though GS’ reaction and ‘will’ is detoxification of ammonia, returning glutamate back to glutamine can be inhibition of glutamine metabolism’s first stage. TRAP1’s binding should be inhibition of GS activity in this manner. The activity assay of GS with and without TRAP1 should be done to confirm whether GS is inhibited or activated by TRAP1.

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References

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Cadoret. A., Ovejero. C., Terris. B., Souil. E., Levy. L., Lamers. WH., Kitajewski. J, Kahn. A., and Perret. C. New targets of beta-catenin signaling in the liver are involved in the glutamine metabolism.

Oncogene. 2002, 21, 8293 – 8301.

Felts, S. J., Owen. BAL., Nguyen, P., Trepel. J., Donner, DB., and Toft, DO. The hsp90-related Protein TRAP1 Is a Mitochondrial Protein with Distinct Functional Properties. The Journal of Biological Chemistry, 2000, 275, 3305-3312.

Kang, BH., Plescia, J., Dohi, T., Rosa, J., Doxsey, SJ., and Altieri, DC., Regulation of Tumor Cell Mitochondrial Homeostasis by an Organelle-Specific Hsp90 Chaperone Network. Cell, 2007, 131, 257–270.

Lobley. G. E., Connella. A., Lomaxa. MA., Browna. DS., Milnea. E., Caldera. AG., and Farninghama.

DAH., Hepatic detoxification of ammonia in the ovine liver: possible consequences for amino acid catabolism. British Journal of Nutrition. 1995, 73, 667-685.

Matthews. GD., Gur. N., Koopman. WJH., Pines. O., and Vardimon. L. Journal of Cell Science, 2009, 123, 351-359.

Mavri-Damelin. D., Damelin. LH., Eaton. S., Rees. M., Selden. C., and Hodgson. HJF., Biotechnol Bioengineering. 2007, 99, 644-51.

Meister. A., Glutathione Metabolism and its Selective Modification, THE JOURNAL OF BIOLOGICAL CHEMISTRY. 1988, 263, 17205-17208,

Rajagopalan. KN., and DeBerardinis. RJ., Role of glutamine in cancer: therapeutic and imaging implications. J Nucl Med. 2011, 52, 1005–1008

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Shivapurkar, N., Reddy, J., Chaudhary, PM., and Gazdar, AF. Apoptosis and Lung Cancer: A Review.

Journal of Cellular Biochemistry. 2003, 88, 885–898.

Takanori Muto, Takayuki Obita, Yoshito Abe, Toshihiro Shodai, Toshiya Endo, and Daisuke Kohda.

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Vander Heiden, MG., Cantley, LC., and Thompson, CB. Understanding the Warburg Effect: The Metabolic Requirements of Cell Proliferation. Science. 2009, 324, 1029-1033.

Vermeulen, Katrien., Van Bockstaele, DR., and Berneman, ZN. The cell cycle: a review of regulation, deregulation and therapeutic targets in cancer. Cell Prolif. 2003, 36, 131–149.

Young. JC., Hoogenrad. NJ., and Hartl. FU. Molecular chaperones Hsp90 and Hsp70 deliver preproteins to the mitochondrial import receptor Tom70. Cell, 2003, 112, 41–50.