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. 2016 Oct;15(10):3107-3125.
doi: 10.1074/mcp.M116.061333. Epub 2016 Aug 8.

Human Sirtuin 2 Localization, Transient Interactions, and Impact on the Proteome Point to Its Role in Intracellular Trafficking

Hanna G Budayeva  1 , Ileana M Cristea  2
Affiliations

Human Sirtuin 2 Localization, Transient Interactions, and Impact on the Proteome Point to Its Role in Intracellular Trafficking

Hanna G Budayeva et al. Mol Cell Proteomics. 2016 Oct.

Abstract

Human sirtuin 2 (SIRT2) is an NAD+-dependent deacetylase that primarily functions in the cytoplasm, where it can regulate α-tubulin acetylation levels. SIRT2 is linked to cancer progression, neurodegeneration, and infection with bacteria or viruses. However, the current knowledge about its interactions and the means through which it exerts its functions has remained limited. Here, we aimed to gain a better understanding of its cellular functions by characterizing SIRT2 subcellular localization, the identity and relative stability of its protein interactions, and its impact on the proteome of primary human fibroblasts. To assess the relative stability of SIRT2 interactions, we used immunoaffinity purification in conjunction with both label-free and metabolic labeling quantitative mass spectrometry. In addition to the expected associations with cytoskeleton proteins, including its known substrate TUBA1A, our results reveal that SIRT2 specifically interacts with proteins functioning in membrane trafficking, secretory processes, and transcriptional regulation. By quantifying their relative stability, we found most interactions to be transient, indicating a dynamic SIRT2 environment. We discover that SIRT2 localizes to the ER-Golgi intermediate compartment (ERGIC), and that this recruitment requires an intact ER-Golgi trafficking pathway. Further expanding these findings, we used microscopy and interaction assays to establish the interaction and coregulation of SIRT2 with liprin-β1 scaffolding protein (PPFiBP1), a protein with roles in focal adhesions disassembly. As SIRT2 functions may be accomplished via interactions, enzymatic activity, and transcriptional regulation, we next assessed the impact of SIRT2 levels on the cellular proteome. SIRT2 knockdown led to changes in the levels of proteins functioning in membrane trafficking, including some of its interaction partners. Altogether, our study expands the knowledge of SIRT2 cytoplasmic functions to define a previously unrecognized involvement in intracellular trafficking pathways, which may contribute to its roles in cellular homeostasis and human diseases.

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Figures

Fig. 1.
Fig. 1.
Experimental design for characterizing SIRT2 interactions in human fibroblasts. A, SIRT2-EGFP is localized to the cytoplasm. MRC5 cells stably expressing SIRT2-EGFP and EGFP were imaged by direct fluorescence microscopy, using DAPI as nuclear marker (blue). Bar size = 25 μm, 63Xmag. B, SIRT2-EGFP localizes similarly to endogenous SIRT2. MRC5 SIRT2-EGFP were imaged by immunofluorescence microscopy using anti-GFP (green) and anti-SIRT2 (red) antibodies, and DAPI nuclear stain (blue). Bar size = 25 μm, 63Xmag. Max Projection. C, SIRT2 mRNA levels in wt, EGFP, and SIRT2-EGFP MRC5 cells. SIRT2 levels were normalized to actin levels across all samples, n = 2. D, SIRT2 protein levels in EGFP and SIRT2-EGFP MRC5 cells. SIRT2-EGFP and endogenous SIRT2 were detected in MRC5 EGFP and SIRT2-EGFP whole-cell lysates using anti-SIRT2 antibody. Histone H4K16ac is a known SIRT2 substrate. Total histone H4 and actin are loading controls. E, SIRT2-EGFP construct is catalytically active. Deacetylase activity of SIRT2-EGFP and EGFP alone was measured upon IP from MRC5 cells using in vitro fluorometric assay. Western blotting using anti-GFP antibody shows equal isolation levels for each protein in the replicates (n = 3). F, Workflow using label-free and metabolic labeling approaches for identifying SIRT2 interactions, their specificity and relative stability. G, Workflow using metabolic labeling for determining changes in the cellular proteome upon shRNA-mediated knockdown of SIRT2.
Fig. 2.
Fig. 2.
SIRT2 interaction network in human fibroblasts. A, Isolation efficiency of SIRT2-EGFP from IP with optimized conditions. SIRT2-EGFP IP was performed from MRC5 cells with optimized lysis buffer using anti-GFP antibody conjugated to magnetic beads via epoxy group. Efficiency of isolation was demonstrated by Western blotting using anti-GFP antibody for detection of SIRT2-EGFP. Soluble - soluble fraction after lysis, Pellet - insoluble fraction, Input - IP input, FT - IP flow through, IP - IP elution, Beads - IP beads fraction after elution. B, Network of SIRT2-interacting partners. Interacting proteins were identified by nLC-MS/MS analysis of SIRT2-EGFP and EGFP control IPs as in Fig. 1F. SAINT algorithm was used to calculate specificity scores based on the number of spectral counts for each protein identified. Proteins passing a SAINT cut-off score of 0.8 were included in the network generated from string-db.com and Cytoscape software. Each node represents an interacting protein, where node color indicates protein enrichment in isolated fraction (NSAF) over its relative enrichment in the human proteome (PAX). Node shape indicates whether the protein is known to be acetylated (diamond) based on uniprot.org records. Protein clusters were assigned based on their known intracellular localizations and functions.
Fig. 3.
Fig. 3.
Analysis of the relative stability of SIRT2 interactions. A, Combination of label-free and metabolic labeling MS-based approaches provides information on specificity and relative stability of SIRT2 interactions in human fibroblasts. B, Comparison of I-DIRT and SAINT scores from SIRT2-EGFP IP experiments. For the I-DIRT-based experiment, SIRT2-EGFP IP was performed upon mixing of MRC5 wt and MRC5 SIRT2-EGFP labeled light (Arg0, Lys0) and heavy (Arg6, Lys6), respectively. I-DIRT scores were calculated as a heavy/(heavy+light) ratio based on precursor peak intensity areas. Each dot represents an interacting protein identified in both experimental approaches. C, Table of interactions identified in SAINT and I-DIRT experiments color-coded based on I-DIRT ratio, as indicated in A. All shown interactions passed SAINT specificity score cutoff unless specified otherwise. Red outline indicates known SIRT2 substrates. D, Representative I-DIRT precursor peak intensity profiles for selected interacting proteins (SFRP1, PPFiBP1, SSR1).
Fig. 4.
Fig. 4.
Mapping SIRT2 interactions to intracellular membrane trafficking pathways. A, SIRT2 interacting proteins are part of major secretory pathways and compartments. Schematic showing proteins identified as interacting partners by SIRT2-EGFP IP (orange) and their known intracellular localization sites. B–C, SIRT2-EGFP is juxtaposed to the nucleus, showing partial colocalization with ER and Golgi. Live MRC5 cells stably expressing SIRT2-EGFP (green) were imaged by confocal microscopy with ER-tracker (B, red) and Golgi-tracker (C, red) fluorescent stains. Bars - 25 μm (D) SIRT2-EGFP colocalizes with ERGIC. MRC5 cells stably expressing SIRT2-EGFP were imaged by immunofluorescence microscopy with anti-GFP (green) and anti-ERGIC53 (red) antibodies. E, Endogenous SIRT2 colocalizes with ERGIC. Wild type MRC5 cells were imaged by immunofluorescence microscopy with anti-SIRT2 (green) and anti-ERGIC53 (red) antibodies. Bars - 50 μm (F) SIRT2-EGFP localization was assessed with Brefeldin A treatment in MRC5 cells stably expressing SIRT2-EGFP. Immunofluorescence microscopy was performed using anti-EGFP (green) and anti-ERGIC53 (red) antibodies. Bars - 25 μm. DAPI is a nuclear marker in B–F.
Fig. 5.
Fig. 5.
SIRT2 interacts and colocalizes with liprin-β1 (PPFiBP1). A, SIRT2 colocalizes with PPFiBP1. SIRT2-EGFP MRC5 cells were imaged by direct fluorescence (SIRT2-EGFP, green) or immunofluorescence microscopy (anti-PPFiBP1, red). Bars - 25 μm. B, PPFiBP1 reciprocally interacts with SIRT2. PPFiBP1 and IgG control IPs were performed in parallel. Western blotting analysis was done using anti-GFP antibody to detect SIRT2-EGFP. Input - 3% (v/v) of IP input fraction, IP - 30% (v/v) of IP elution fraction. C, PPFiBP1 localization was assessed with or without Brefeldin A treatment in MRC5 cells stably expressing SIRT2-EGFP. Immunofluorescence microscopy was performed using anti-EGFP (green) and anti-PPFiBP1 (red) antibodies. DAPI is a nuclear stain. Bars - 50 μm.
Fig. 6.
Fig. 6.
SIRT2 impact on protein levels of membrane trafficking proteins in human fibroblasts. A, SIRT2 mRNA levels upon shRNA-mediated knockdown in MRC5 cells. MRC5 cells stably expressing nontargeting shRNA (shCtrl) or shSIRT2 were subjected to mRNA extraction and analysis of SIRT2 mRNA levels by qPCR. Β-Actin was used as endogenous control, n = 3. B, SIRT2 protein levels upon shRNA-mediated knockdown in MRC5 cells. Total endogenous SIRT2 levels were measured in MRC5 cells stably expressing shCtrl or shSIRT2 by Western blotting using anti-SIRT2 and anti-β-actin antibody as loading control. C, Changes in total protein levels upon SIRT2 KD in MRC5 cells from two replicate experiments. MRC5 shCtrl and shSIRT2 were differentially labeled by SILAC and total protein levels were assessed by mass spectrometry. Each dot represents quantified protein and its color indicates local point density. D, Table of GOBP categories with top annotation enrichment scores. Data set of changes in protein levels upon SIRT2 KD was analyzed in Perseus using 2D annotation enrichment algorithm based on two replicate experiments. GOBP enrichment scores were similar between replicates and one representative score from the two replicates is indicated. E, Changes in individual protein levels upon SIRT2 KD in MRC5 cells.
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