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

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.

RTN4B‐mediated suppression of Sirtuin 2 activity ameliorates β‐amyloid pathology and cognitive impairment in Alzheimer's disease mouse model

“…Then, we further locked five candidates that may serve as SIRT2 targets in AD progression by informatics analysis and finally identified RTN4 as the most impressive candidate for that it may interact with BACE1 (He et al, 2004; Murayama et al, 2006). Besides, a previous study that using metabolic labeling quantitative MS also suggested that RTN4 may transiently target SIRT2 (Budayeva & Cristea, 2016), and this possible interaction was also identified in other proteomic studies (Huttlin et al, 2015), supporting our results. RTN4, also known as Nogo, is one member of the RTN family and has mainly three isoforms (RTN4A, RTN4B, RTN4C), which are abundant in the nervous system (Chen et al, 2000).…”

SIRT1 activation with neuroheal is neuroprotective but SIRT2 inhibition with AK7 is detrimental for disconnected motoneurons

“…2a ). SIRT2 appeared at a characteristic location probably corresponding to the ER–Golgi intermediate compartment 23 , and it was present at lower levels in axotomized MNs at 7 dpi with respect to the control (Fig. 2b ).…”

Active nuclear import of the deacetylase Sirtuin-2 is controlled by its C-terminus and importins

“…To gain further insight into the functional roles of the newly identified SIRT2 interactors, gene ontology term enrichment analysis was performed using PANTHER 21 . The most enriched GO terms for biological processes included tricarboxylic acid cycle 22 , vesicle-mediated transport 8 and fatty acid catabolic processes 23 (Table S2). These are consistent with the growing body of reports which implicate SIRT2 as a key regulator of metabolic processes within the cell 7 .…”