Molecular Cell, Volume 50 Supplemental Information

SIRT5-Mediated Lysine Desuccinylation Impacts Diverse Metabolic Pathways Jeongsoon Park, Yue Chen, Daniel X. Tishkoff, Chao Peng, Minjia Tan, Lunzhai Dai, Zhongyu Xie, Yi Zhang, Bernadette M. M. Zwaans, Mary E. Skinner, David B. Lombard, and Yingming Zhao

Supplemental Experimental Procedures

Generation of Sirt5+/+ and Sirt5-/- MEFs

Chimeric mice were generated by injection of targeted ES clones into C57BL6/J

blastocysts. Male chimeras were mated with 129Sv females to generate F1

heterozygous mice, which were interbred to generate homozygous KO mice. MEFs

were generated from day 13.5 embryos by standard methods. Sirt5+/+ (WT) and Sirt5-/-

(KO) MEFs were immortalized by infecting with a retroviral vector SSR#41 that

expresses SV40 large T antigen flanked with FRT sites. The infected cells were

subsequently selected in DMEM media containing 0.3 mg/mL hygromycin B (Invitrogen,

Carlsbad, CA, USA) for 7–10 days.

Stable Isotope Labeling of Sirt5 KO and WT MEFs

Sirt5 KO and WT MEFs were labeled with light ([U-12C614N2]-L-lysine) and heavy ([U-

13C615N2]-L-lysine) lysine using a SILAC protein quantification kit (Invitrogen, Carlsbad,

CA) according to manufacturer’s instructions. The two cell lines were grown for more

than 6 generations in SILAC DMEM media supplemented with dialyzed fetal bovine

serum (FBS) and either “light” lysine or “heavy” lysine to achieve more than 99%

labeling efficiency.

2

Preparation of MEF Cell Lysate

The cells were washed twice with pre-chilled PBS and lysed with SDS sample buffer (20

mM Tris pH 6.8, 2% SDS, 5% β-mercaptoethanol, 10% glycerol, and 25 mM

nicotinamide). The cell lysates from “heavy” and “light” cells were clarified by

centrifugation for 10 min at 20,000xg, respectively. Equivalent amounts of protein

lysates from two pools of cells were combined and digested with trypsin for high-pH

reverse-phase HPLC fractionation.

Peptide Fractionation with High-pH Reverse-Phase Chromatography

HPLC buffer A (10 mM ammonium formate in water, pH 7.8) and HPLC buffer B (10 mM

ammonium formate in 90% acetonitrile and 10% water, pH 7.8) were used for high-pH

reverse-phase fractionation of peptides. The reverse-phase column (Phenomenex,

Luna C18 10 mm x 250 mm, 5 µm particle, 100 Å pore size) was equilibrated with 2%

HPLC buffer B. The peptide mixture of interest in HPLC buffer A was loaded onto the

HPLC column and eluted with linear gradient of 2% to 30% HPLC buffer B in 40 min,

30% B-90% B in 15 min at a constant flow rate of 4 mL/min using Shimadzu preparative

HPLC. Seventy fractions were collected and combined equally into 20 final fractions.

The fractions were dried in a SpeedVac prior to immunoaffinity enrichment.

Preparation of Mouse Liver Lysate and Protein Fractionation

Four-month old male Sirt5 KO mice (Lombard et al., 2007; Nakagawa et al., 2009) were

anesthetized with isoflurane, and the blood in the liver was removed by perfusion with

3

ice cold NaCl (0.9%) for 1 min. Livers were collected immediately and homogenized in a

glass dounce tissue grinder in NETN lysis buffer (100 mM NaCl, 20 mM Tris-Cl pH 8.0,

0.5 mM EDTA, 1.0 % (v/v) Nonidet P-40 (NP40), and 20 mM nicotinamide). The tissue

homogenate was sonicated on ice for 4 x 15 sec with 1 min cooling intervals. The lysate

was centrifuged at 20,000xg and the pellet was saved as an independent fraction for

trypsin digestion. Proteins in the supernatant were fractionated by HPLC using a

polyCAT/WAX column following a previously described method (Chen et al., 2012). A

total of 20 fractions were collected. The proteins in each fraction were precipitated with

trichloroacetic acid (TCA) and then in-solution digested by trypsin as previously

described prior to immunoaffinity purification (Kim et al., 2006a).

PTM-Specific Immunoaffinity Enrichment

Enrichment of Ksucc peptides was performed as previously described (Kim et al.,

2006b). Briefly, the tryptic peptides were incubated with 50 μl antibody-immobilized

beads (PTM Biolabs, Chicago, IL) at 4 °C for 6 hours with gentle rotation. After

incubation, the beads were carefully washed three times with NETN buffer (50 mM

Tris•HCl [pH 8.0], 100 mM NaCl, 1 mM EDTA, 0.5% NP40), twice with ETN buffer (50

mM Tris•HCl [pH 8.0], 100 mM NaCl, 1 mM EDTA) and once with water. The bound

peptides were eluted from the beads by washing three times with 30 μl of 0.1 M glycine

solution (pH = 2.5). Eluates were combined and dried in a SpeedVac. Quantitative

proteomics analysis of Kac peptides using SILAC-based mass spectrometric analysis

was carried out likewise using anti-Kac antibody-immobilized beads (PTM Biolabs,

Chicago, IL).

4

HPLC-MS/MS Analysis of Tryptic Peptides

The tryptic digest were dissolved in HPLC solvent A (0.1% formic acid in water, v/v) and

injected into an EASY-nLC 1000 HPLC system (Thermo Fisher Scientific, Odense,

Denmark) equipped with an in-house made fused silica emitter (10 cm length, 360 μm

OD x 75 μm ID) packed with Jupiter C12 resin (4 μm particle size, 90 Å pore size,

Phenomenex). All the digested samples were eluted with a 60 min gradient from 5%

solvent B (10%water/ 90%ACN/0.1% formic acid, v/v/v) to 80% solvent B at a flow rate

of 200 nL/min. The eluates from the RP-HPLC column were ionized by a Proxeon

nanoESI ion source and introduced into an Orbitrap Elite mass spectrometer system.

Full MS spectra from m/z 350 to 1700 were acquired in the Orbitrap mass analyzer at a

resolution of 240,000 at 400 m/z. Lock mass at m/z 536.165369 was enabled for

internal mass calibration for the full MS scan. The full MS scans were followed by data-

dependent MS/MS fragmentation of the 15 most intense peaks in the dual linear ion trap

mass analyzer in rapid scan mode with a normalized collision-induced dissociation (CID)

energy of 35%. A forty-second dynamic exclusion time with a repeat count of 1 and

±10ppm exclusion window was used for MS/MS analysis.

SILAC-Based Quantification Analysis

Protein and PTM site identification and quantifications were performed by Maxquant

with integrated Andromeda search engine (v. 1.3.0.5) (Cox and Mann, 2008; Cox et al.,

2011). For each MS/MS spectra, the top 6 most intense peaks in every 100 Da window

were extracted. Then, tandem mass spectra were searched against Uniprot mouse

5

reference protein database concatenated with reverse decoy database and protein

sequences of common contaminants. Trypsin was specified as cleavage enzyme with 1

missing cleavage for protein quantification and 2 missing cleavages for PTM site

quantification with the maximum of 3 labeled amino acids per peptides. Mass error was

set to 6 ppm for precursor ions and 0.5 Da for fragment ions. Carbamyiodomethylation

on Cys was specified as fixed modification and oxidation on Met, acetylation on Lys,

succinylation on Lys and acetylation on protein N-terminal were specified as variable

modifications. False discovery rate (FDR) thresholds for protein, peptide and

modification site were specified at 0.01. Minimum peptide length was set at 7. In-house

developed scripts were applied to remove Lys succinylation site identifications with

Andromeda score less than 40 or site localization probability less than 0.75. In addition,

the scripts also removed site identifications from reverse or contaminant protein

sequences, as well as Lys acetylation or succinylation sites assigned to the peptide C-

terminal, unless the peptide C-terminal was also the corresponding protein C-terminal.

Estimation of Absolute Stoichiometry of Lys Succinylation Sites

We modified a previous reported algorithm to estimate absolute stoichiometry of Ksucc

sites quantified in WT and Sirt5 KO MEF cells (Olsen et al., 2010). For each Ksucc site,

the method requires the successful quantification of a Ksucc site, as well its

corresponding protein and unmodified peptide. We required that all proteins were

quantified considering any peptides but succinylated peptides. For each Ksucc peptide,

we define the unmodified form as the longest and completely tryptically digested portion

of the Ksucc peptide and we required that the unmodified peptide not have any other

6

variable modification or missing cleavages. Un-normalized SILAC KO/WT quantification

ratios of Ksucc peptides, corresponding proteins and unmodified peptides, were then

analyzed following the previously reported algorithm (Olsen et al., 2010).

Functional Annotation Analysis

Enrichment analysis for Gene Ontology (Ashburner et al., 2000), KEGG pathway

(Kanehisa and Goto, 2000) and Disease Ontology (Schriml et al., 2012) were performed

with GO-Elite package (Zambon et al., 2012). Enrichment analysis for PFAM domain

(Finn et al., 2008) was performed using a hypergeometric test in GOstats package

(Falcon and Gentleman, 2007) in R. All p values were adjusted with a Benjamini-

Hochberg false discovery rate with a cutoff rate of 1% (Benjamini and Hochberg, 1995).

Protein Complex Analysis

Manually curated CORUM protein complex database for all mammals was used for

protein complex analysis (Ruepp et al., 2008). Overrepresented complexes were

identified using hypergeometric test with Benjamini-Hochberg corrected false discover

rate of less than 1%.

Protein-Protein Interaction Network Analysis

All interactions for mouse with scores above 700 in the STRING database were used for

protein-protein interaction analysis. The network was visualized in Cytoscape (v.3.0

beta) (Shannon et al., 2003) and highly-connected clusters were identified by MCODE

plug-in (Bader and Hogue, 2003).

7

In Vitro Desuccinylation Assay

Prior to use, porcine heart PDC (Sigma; P7032) was further purified by pelleting at

135,000xg for 2 h in a buffer containing 100 mM KH2PO4 pH 7.5, 0.05% Lauryl

Maltoside, 2.5 mM EDTA, and 30% glycerol. Desuccinylation reactions were carried out

on 20.8 μg purified PDC in a final volume of 80 μl in reaction buffer consisting of 25 mM

Tris-Cl pH 8.0, 200 mM NaCl, 5 mM KCl, 1 mM MgCl2, 0.1% PEG 8000, and 3.125 mM

NAD+ at 37°C for 1 hr. Where indicated, 24 μg of WT or mutant SIRT5 was added.

Following desuccinylation, 15 μl of the reaction was used for immunoblot analysis with

Ksucc antibody (PTM BioLab Co. Ltd, #PTM-401) and 50 μl of the reaction was

analyzed by PDC AAT activity assay.

Sirt5 KD Cell Generation

Stable Sirt5 KD 293T cell lines were established by lentiviral transduction of Sirt5

shRNAs in the pLKO lentiviral backbone (Sigma oligo ID#sTCN0000018546 and

TCN0000018547/cat # RHS4533-NM12241) and a non-silencing shRNA as a control.

Transduced cells were selected in 4 μg/ml puromycin until all uninfected control cells

were dead.

Subcellular Fractionation

293T cells were collected by centrifuging at 500xg for 5 min at 4°C. Cells were gently

homogenized in hypotonic buffer (10 mM Hepes pH 7.9, 10 mM KCl, 0.1 mM EDTA)

containing protease inhibitors (0.5 mM PMSF and protease inhibitor complete (Roche))

8

for 15 min on ice with the use of a dounce homogenizer, and then centrifuged at 800xg

for 5 min at 4°C to obtain the pellet (nuclear fraction). The supernatant was centrifuged

at 6,200xg for 10 min at 4°C to pellet mitochondria. A portion of the remaining

supernatant was used for immunoblot as the cytosolic fraction. The mitochondrial pellet

was solubilized with 1% sodium dodecylmaltoside in PBS pH 7.4 after two washes and

centrifugation at 100,000xg for 15 min at 4°C. For immunoblot, equal protein amounts of

each fraction was loaded as assessed by DC assay (Bio-rad).

For tissue fractionation, livers were rinsed in cold PBS and then minced on ice and

homogenized in LHM containing a protease inhibitor cocktail (Roche), 500 nM

trichostatin A (TSA), and 10 mM nicotinamide using a chilled glass dounce homogenizer.

Homogenates were centrifuged for 10 min at 800xg at 4°C, and the resulting pellets

were extracted to obtain the nuclear fraction. The nuclear fraction was resuspended in

0.5% NP40 in 50 mM Tris-Cl (pH 7.5) and 150 mM NaCl, then again pelleted and

extracted with 20 mM Tris-Cl (pH 7.9), 25% glycerol, 0.42 M NaCl, 1.5 mM MgCl2,

0.2 mM EDTA, 0.1 mM PMSF and 0.5 mM DTT for 20 min, and centrifuged at 20,000xg

for 30 min. The original supernatants were centrifuged at 6,200xg for 10 min, and the

resulting pellet was used as the mitochondrial fraction, and the supernatant was used

as the cytosolic fraction.

SIRT5 Immunofluorescence

For immunofluorescent detection of SIRT5, 293T cells grown on glass cover slips were

preincubated for 15 min with 500 nM MitoTracker CMRox (Molecular Probes) and then

fixed with 4% paraformaldehyde for 15 minutes at room temperature. After washing with

9

phosphate-buffered saline (PBS), pH 7.4, cells were incubated with 0.1% Triton X-100

in PBS for 10 min. Following blocking with 2% goat serum, cells were incubated at 37°C

with 1:200 diluted rabbit SIRT5 antibody (HPA022002, Sigma) or rabbit IgG as a

negative control for 1 hour. Following 3 washes with PBS, anti-rabbit Alexa fluor 488-

conjugated secondary antibody (Molecular Probes) was used for detection. For nuclear

staining, cells were incubated with 500 nM DAPI for 15 minutes at room temperature

before mounting in Prolong Gold Antifade (Invitrogen). Cells were visualized using a

FV300 BX61 laser-scanning confocal fluorescence microscope 300 (Olympus, Melville,

NY). Images were acquired with FluoView analyzer.

PDC INT Activity

For measurement of PDC activity in cells by INT assay (Schwab et al., 2005), cells were

harvested with 0.05% trypsin–EDTA, washed twice with PBS, and subsequently

centrifuged (500xg). Cells were then suspended at a final protein concentration of 1

mg/ml in PBS. After PDC activation with DCA (5 mM) for 15 minutes at 37°C where

indicated (Fouque et al., 2003), cells were washed once with PBS, and the cell pellet

was resuspended in 3 ml of buffer containing 250 mM sucrose, 1 mM EGTA, and 10

mM Hepes, pH 7.5 and homogenized by 20 strokes in a tight-fitting dounce

homogenizer on ice. After centrifugation at 13,000xg for 5 minutes at 4°C, the

supernatant was discarded. Pellets were then subject to 3 rounds of freeze-thaw in LN2

and 25°C water. Cells were then resuspended at ~4 mg/ml in a buffer consisting of

0.1% Triton X-100, 1 mM CaCl2, 5 mM MgCl2, 50 mM KCl, 250 mM sucrose, and 20

mM Tris-HCl pH 7.5. Between 100 μg-1 mg of sample was used per 1.1 ml of reaction

master mix consisting of 5 mM L-carnitine, 2.5 mM NAD+, 0.2 mM TPP, 0.1 mM CoA,

10

0.5 mM pyruvate, 0.1% Triton X-100, 1 mM MgCl2, 0.1% BSA, 0.6 μM p-

iodonitrotetrazolium chloride (INT), and 6.5 μM phenazine methylsufate (PMS) in 50 mM

potassium phosphate buffer, pH 7.5 (Hinman and Blass, 1981). Assays were performed

at 25°C in a SpectraMax plus plate reader; the increase in absorbance at 500 nm was

monitored, attributable to reduction of INT, the final electron acceptor (extinction

coefficient 12.4 mM-1 cm-1).

SDH Activity Assay

For determination of SDH enzymatic activity, Sirt5 KD and control 293T cell

mitochondria were prepared and purified (Graham, 2001). Solubilization of purified

mitochondria with 1% of n-dodecyl β-D-maltoside and separation of mitochondrial

protein complexes by sucrose density gradient centrifugation were performed as

previously described (Taylor et al., 2002; Taylor et al., 2003). SDH activity was

measured by adding an assay solution containing 10 mM Tris-HCl pH 7.4, 10 mM

KH2PO4, 250 mM sucrose, 1 mM EGTA, 20 mM succinate, 2 mM KCN, 50 μM

dichlorophenolindophenol (DCIP), and 50 M ubiquinone-2 with 10-20 μg of

mitochondrial lysate. The reduction of DCIP was measured at 600 nm in a SpectraMax

plate reader in kinetic mode at room temperature for 30 min. Calculations were based

on the extinction coefficient of 21 mM-1 cm-1 for DCIP (Nadanaciva et al., 2007)

Oxygen Consumption Assay in Cells

For measurements of O2 consumption rate in cells, ~5x106 cells were permeabilized in

0.01% digitonin and resuspended in respiration buffer (20 mM Hepes, pH 7.1, 250 mM

11

sucrose, 2 mM Kpi, 10 mM MgCl2, and 1.0 mM ADP). Measurements were carried out

using a Clark-type oxygen electrode (Hansatech Ltd.) by sequential addition of

substrates and inhibitors. The substrate concentrations were as follows: malate,

pyruvate, succinate, all 5 mM; ascorbate, 10 mM; N,N, N’, N’-tetramethyl-p-

phenylenediamine (TMPD), 0.2 mM; rotenone, 1 �M; 6-fluoropyruvate, 5 mM; antimycin

A, 3 μg/ml; KCN 2 mM. Reduction of oxygen was calculated by normalizing to actual

cell number in each (Hofhaus et al., 1996).

Mitochondrial Respiration

Respiration of isolated mitochondria was measured using a Seahorse XF-24 Analyzer

(Seahorse Bioscience). Mitochondria were diluted to 0.2 mg/ml in BSA-free LM (70 mM

sucrose, 220 mM mannitol, 1 mM EGTA, 2 mM Hepes, 1% w/v defatted BSA, pH 7.2)

(Mailloux et al., 2013), and 50 μl of suspension (10 μg mitochondria, except for

Ascorbate/TMPD where 5 μg were used) was then loaded into Seahorse tissue culture

(TC) plate wells. TC plates were centrifuged at 2,000xg for 10 min at 4 °C to attach

mitochondria to the plastic. For bioenergetic determinations, the oxygen consumption

rate (OCR) was first determined under state 2 conditions (substrate only: 10 mM

pyruvate and 5 mM malate, or 5 mM succinate and 2 μM rotenone, or 5 mM glutamate

and 5 mM malate, or 1 mM TMPD and 100 mM ascorbate. ADP (0.1 mM), oligomycin

(2.5 μg/ml), FCCP (2 μM), and 5 mM fluoropyruvate or 2 μM rotenone or antimycin A (4

μM), or sodium azide were then sequentially injected to test state 3, state 4 (proton

leak-dependent respiration), maximal respiration, and respiration independent of the

respiratory chain, respectively.

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siRNA Transfection

Smartpool siRNAs targeting human Sdhc (L-011385-00-0005), Sdhd (L006305-00-

0005), and the non-targeting control (S-001810-10-05) were purchased from

Dharmacon Research (Lafayette, Colorado), and transfection of siRNAs was performed

according to manufacturer's instructions. Briefly, 293T cells were trypsinized and plated

in 6-well dishes at 2.5x104 cells per well for 12−16 h prior to transfection. Cationic lipid

complexes, prepared by incubating 200 pmol of the indicated siRNA with 2μl

DharmaFECT (Dharmacon) in 100 μl DMEM (Gibco-Invitrogen) for 20 min, were added

to the wells in a final volume of 1 ml. After 24 hours, cells were passaged and harvested

at 48, 72, and 96 hours for Ksucc immunoblot.

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Figure S1. Sirt5 KO Results in Global Increase of Lys Succinylation in Mouse Liver

Tissue and MEFs, Related to Figure 1A

(A) Succinylation in mouse liver tissue.

(B) Succinylation in MEFs.

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Figure S2A. Immunoblot Analysis of Ksucc Levels in Liver and Kidney Whole-Cell

Extracts and Mitochondria of ~8-Week-Old 129SvE Male and Female Mice Fed ad lib or

Fasted for 48 hr, Related to Figure 1B

15

Figure S2B

16

Figure S2C

17

Figure S2D

18

Figure S2E

19

Figure S2F

20

Figure S2G

21

Figure S2H

22

Figure S3. Related to Figure 2A

(A) Histograms showing distributions of Lys acetylation and succinylation peptides

against their dynamic changes between Sirt5 KO and WT MEFs.

(B) Scatter plots showing the dynamics of protein abundances in Sirt5 KO. Data points

were colored based on absolute z scores of the protein SILAC KO/WT Log2 ratios – red

points having z values less than 1, yellow points having z values between 1 and 2,

green points having z values between 2 and 3, and blue points having z values larger

than 3.

23

Figure S4. Sequence Logo Representation of Significant Motifs Identified among Ksucc

Sites, Related to Figure 4A

24

Figure S5A. Illustrated Examples of KEGG Pathways Enriched with Ksucc Proteins,

Related to Figure 4C

Red borders designate proteins with Lys succinylation.

(A) Val-Leu-Ile degradation pathway.

25

Figure S5B. Citrate Cycle.

26

Figure S5C. Fatty Acid Metabolism

27

A

B

Figure S6. Kinetics of Lys Succinylation, Related to Figure 2B

Hela cells were pulse-labeled with 50 mM D4-succinate for 0, 8, and 24 h. Collected

cells were lysed and proteins were digested by trypsin prior to immunoaffinity

purification of Ksucc peptides by pan anti-Ksucc antibody. Labeling ratios were

28

calculated based on intensity ratios of heavy (D4) form to light form (H4) of each Ksucc

peptide from every time point. (A) Clustering of labeling ratios from 15 peptides

identified in both heavy and light forms at all three timepoints. (B) Linear regression

analysis for the Ksucc peptides in cluster 2 from clustering analysis in (A) to calculate

the percentage rate of Lys D4-succinylation incorporation (left panel) and the

percentage rate of the Lys H4-succinylation loss (right panel). The error bars represent

the standard error of means (SEM) for all labeling ratios in each time point group.

29

A

B

C

30

Figure S7. SDH Inhibition Does Not Greatly Impact Lys Succinylation, Related to Figure

6A

(A and B) WT or SIRT5-deficent mouse fibroblasts were treated with the SDHA

inhibitors 3-NPA or malonate as indicated, and Ksucc levels were evaluated by

immunoblot.

(C) 293T cells were transfected with siRNA smartpools directed against either Sdhc or

Sdhd. Sdhc was efficiently inhibited; however Sdhd KD was unsuccessful. Ksucc levels

were evaluated by immunoblot

31

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