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

Hanna G Budayeva,Ileana M Cristea

doi:10.1074/mcp.m116.061333

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.

Highlights

Human sirtuin 2 (SIRT2)1 is one of seven NADϩ-dependent deacylases (SIRT1–7) that were originally discovered as homologues of S
In agreement with a moderate level of overexpression, we observed that SIRT2 mRNA levels were two times higher in the population of SIRT2-EGFP cells than in the wt or EGFP control MRC5 cells, indicating that at the mRNA level SIRT2-EGFP is present at relatively similar levels to endogenous SIRT2 (Fig. 1A)
SIRT2 Role in Membrane Trafficking and Implications in Cellular Homeostasis and Human Disease—This study integrated microscopy, protein interaction, knockdown, and functional assays to investigate the roles of SIRT2 in primary human fibroblasts

Summary

Introduction

Human sirtuin 2 (SIRT2) is one of seven NADϩ-dependent deacylases (SIRT1–7) that were originally discovered as homologues of S. SIRT7 was found to play a role in RNA Pol I-dependent transcription through its interactions with the B-WICH chromatin remodeling complex [30] Both SIRT1 and SIRT2 were shown to modulate gene expression through deacetylation of several transcription factors, including nuclear factor ␬〉 (NF␬〉) [31, 32], p53 [33, 34], forkhead box protein O3 (FOXO3A) [35, 36], and coactivator ␤-catenin [37, 38]. The neuroprotective effects of SIRT2 inhibition in Huntington’s disease was associated with the SIRT2-dependent regulation of the SREBP-2 transcription factor trafficking from the endoplasmic reticulum to the nucleus [39] These studies highlight the importance of continuing the evaluation of sirtuin impact on gene expression and total protein levels

Objectives

Results

Conclusion