Subcellular localization and mitotic interactome analyses identify SIRT4 as a centrosomally localized and microtubule associated protein

Abstract

The stress-inducible and senescence-associated tumor suppressor SIRT4, a member of the family of mitochondrial sirtuins (SIRT3, SIRT4, and SIRT5), regulates bioenergetics and metabolism via NAD + -dependent enzymatic activities. Next to the known mitochondrial location, we found that a fraction of endogenous or ectopically expressed SIRT4, but not SIRT3, is located at the mitotic spindle apparatus in the cytosol. Confocal spinning disk microscopy revealed that SIRT4 localizes during the cell cycle dynamically at centrosomes with an intensity peak in G2 and early mitosis.Moreover, SIRT4 binds to microtubules and interacts with structural (α,β-tubulin, -tubulin, TUBGCP2, TUBGCP3) and regulatory (HDAC6) microtubule components as detected by coimmunoprecipitation and mass spectrometric analyses of the mitotic SIRT4 interactome.Overexpression of SIRT4 resulted in a pronounced decrease of acetylated α-tubulin (K40) associated with altered microtubule dynamics in mitotic cells. SIRT4 or the N-terminally truncated variant SIRT4(ΔN28), which is unable to translocate into mitochondria, delayed mitotic progression and reduced cell proliferation. This study extends the functional roles of SIRT4 beyond mitochondrial metabolism, and suggests that SIRT4 acts as a novel centrosomal / microtubule-associated protein in the regulation of cell cycle progression. Thus, stress-induced SIRT4 may exert its role as tumor suppressor through mitochondrial as well as extramitochondrial functions, the latter associated with its localization at the mitotic spindle apparatus.

Research Progress of Sirtuin4 in Cancer

“…Importantly, the N-terminally truncated variant SIRT4 (ΔN28), which cannot translocate into mitochondria, delayed mitotic progression and reduced cell proliferation. Hence, SIRT4 may exert its anti-proliferative role through mitochondrial/metabolism-dependent and mitochondria-independent functions ( 30 ).…”

Viewing teratogens through the lens of nicotinamide adenine dinucleotide (NAD+)

“…Also, NAD+ and SIRT3, a mitochondrial NAD+ dependent sirtuin, have been shown to control microtubule dynamics and reduce susceptibility to antimicrotubule agents such as vinblastine and colchicine (Harkcom et al, 2014). Additionally, SIRT4, another mitochondrial NAD+ dependent sirtuin, acts as a novel centrosomal/microtubule‐associated protein in the regulation of cell cycle progression (Bergmann et al, 2020).…”

Identification of SIRT4 as a novel paralog-specific interactor and candidate suppressor of C-RAF kinase in MAPK signaling

“…The beads were mixed with Laemmli loading buffer and co-immunoprecipitation of SIRT4-Flag and endogenous C-RAF proteins was analyzed by SDS-PAGE and immunoblotting. Co-immunoprecipitation of SIRT4-eGFP and endogenous C-RAF using the anti-eGFP nanobody protocol was performed essentially as previously described (33).…”

“…In particular, SIRT4 inhibits, as a tumor suppressor, the metabolic gate-keepers pyruvate dehydrogenase (PDH) and glutamate dehydrogenase (GDH) (31,32), with particular significance for the regulation of glutamine metabolism in tumor cells. Recent findings uncovered novel extra-mitochondrial roles of SIRT4 in microtubule dynamics and regulation of mitotic cell cycle progression, WNT/-Catenin and Hippo signaling, and SNARE complex formation required for autophagosome-lysosome fusion (33)(34)(35)(36). Interestingly, proteomic analysis of the SIRT4 interactome identified C-RAF as a potential binding partner of SIRT4, indicating a novel role of SIRT4 in the regulation of the RAF-MAPK signaling pathway (33).…”

“…Recent findings uncovered novel extra-mitochondrial roles of SIRT4 in microtubule dynamics and regulation of mitotic cell cycle progression, WNT/-Catenin and Hippo signaling, and SNARE complex formation required for autophagosome-lysosome fusion (33)(34)(35)(36). Interestingly, proteomic analysis of the SIRT4 interactome identified C-RAF as a potential binding partner of SIRT4, indicating a novel role of SIRT4 in the regulation of the RAF-MAPK signaling pathway (33). Consistent with this idea, recent studies have demonstrated that (i) the tumor suppressor SIRT4 is downregulated in most human solid tumor types and cell lines (37)(38)(39), and (ii) ectopic expression of SIRT4 downregulates pERK1/2 levels and hence inhibits MAPK signaling and cell proliferation (37)(38)(39)(40)(41)(42).…”