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TBC/RABGAPs are negative regulators of RABs that carry a conserved TBC domain. In addition to their roles in intracellular trafficking, they have recently emerged as integrators of signalling between RABs and other small GTPases, and they are frequently dysregulated in disease. The Tre2–Bub2–Cdc16 (TBC) domain-containing RAB-specific GTPase-activating proteins (TBC/RABGAPs) are characterized by the presence of highly conserved TBC domains and act as negative regulators of RABs. The importance of TBC/RABGAPs in the regulation of specific intracellular trafficking routes is now emerging, as is their role in different diseases. Importantly, TBC/RABGAPs act as key regulatory nodes, integrating signalling between RABs and other small GTPases and ensuring the appropriate retrieval, transport and delivery of different intracellular vesicles.

The sirtuin SIRT4 has been implicated in the control of autophagy and mitochondrial quality control via mitophagy. However, the role of SIRT4 in regulating autophagy/mitophagy induced by different stressors is unclear. Here, we show that cells expressing SIRT4(H161Y), a catalytically inactive, dominant-negative mutant of SIRT4, fail to upregulate LC3B-II. These cells also exhibit a reduced autophagic flux upon treatment with different inducers of mitophagy/autophagy, that is, CoCl -triggered pseudohypoxia, CCCP (carbonyl cyanide 3-chlorophenylhydrazone)/oligomycin-mediated respiratory chain inhibition, or rapamycin treatment. Interestingly, SIRT4(H161Y) expression upregulated protein levels of HDAC6, which is involved in mitochondrial trafficking and autophagosome-lysosome fusion, and inhibited the conversion of OPA1-L to OPA1-S, which is associated with increased mitochondrial fusion and decreased mitophagy. Both HDAC6 and OPA1 are SIRT4 interactors. However, the pharmacological inhibition of HDAC6 using Tubacin or of OPA1 using MYLS22 did not restore the stress-induced upregulation of LC3B-II levels upon autophagy/mitophagy treatment in SIRT4(H161Y)-expressing cells. Remarkably, inhibition of autophagosome-lysosome fusion and thus disruption of late autophagic flux by BafA1 treatment also failed to restore LC3B-II levels upon autophagy/mitophagy treatment, suggesting an inhibitory effect of SIRT4(H161Y) on the initiation/early phase of autophagy. Consistent with this, we demonstrate that SIRT4(H161Y) promotes the phosphorylation of ULK1 at S638 and S758 (mTORC1 targets), both of which mediate an important inhibitory regulation of autophagy initiation. Thus, our data suggest a positive regulatory function of SIRT4 in the ULK1-dependent early regulation/initiation of stress-induced autophagic flux, presumably via modulation of AMPK/mTORC1 signaling.

The sirtuin SIRT4 has been implicated in the control of autophagy and mitochondrial quality control via mitophagy. However, the role of SIRT4 in regulating autophagy/mitophagy induced by different stressors is unclear. Here, we show that cells expressing SIRT4(H161Y), a catalytically inactive, dominant‐negative mutant of SIRT4, fail to upregulate LC3B‐II. These cells also exhibit a reduced autophagic flux upon treatment with different inducers of mitophagy/autophagy, that is, CoCl 2 ‐triggered pseudohypoxia, CCCP (carbonyl cyanide 3‐chlorophenylhydrazone)/oligomycin‐mediated respiratory chain inhibition, or rapamycin treatment. Interestingly, SIRT4(H161Y) expression upregulated protein levels of HDAC6, which is involved in mitochondrial trafficking and autophagosome‐lysosome fusion, and inhibited the conversion of OPA1‐L to OPA1‐S, which is associated with increased mitochondrial fusion and decreased mitophagy. Both HDAC6 and OPA1 are SIRT4 interactors. However, the pharmacological inhibition of HDAC6 using Tubacin or of OPA1 using MYLS22 did not restore the stress‐induced upregulation of LC3B‐II levels upon autophagy/mitophagy treatment in SIRT4(H161Y)‐expressing cells. Remarkably, inhibition of autophagosome–lysosome fusion and thus disruption of late autophagic flux by BafA1 treatment also failed to restore LC3B‐II levels upon autophagy/mitophagy treatment, suggesting an inhibitory effect of SIRT4(H161Y) on the initiation/early phase of autophagy. Consistent with this, we demonstrate that SIRT4(H161Y) promotes the phosphorylation of ULK1 at S638 and S758 (mTORC1 targets), both of which mediate an important inhibitory regulation of autophagy initiation. Thus, our data suggest a positive regulatory function of SIRT4 in the ULK1‐dependent early regulation/initiation of stress‐induced autophagic flux, presumably via modulation of AMPK/mTORC1 signaling.

Unlike the α subunits of heterotrimeric guanosine triphosphate (GTP)-binding proteins, Ras-related GTP-binding proteins have hitherto been considered not to bind or become activated by tetrafluoroaluminate (AlF$_4^-$). However, the product of the proto-oncogene ras in its guanosine diphosphate (GDP)-bound form interacted with AlF$_4^-$ in the presence of stoichiometric amounts of either of the guanosine triphosphatase (GTPase)-activating proteins (GAPs) p120$^{GAP}$ and neurofibromin. Neither oncogenic Ras nor a GAP mutant without catalytic activity produced such a complex. Together with the finding that the Ras-binding domain of the protein kinase c-Raf, whose binding site on Ras overlaps that of the GAPs, did not induce formation of such a complex, this result suggests that GAP and neurofibromin stabilize the transition state of the GTPase reaction of Ras.

RAS-RELATED GTP-binding proteins function as molecular switches which cycle between GTP-bound 'on'- and GDP-bound 'off'-states 1 . GTP hydrolysis is the common timing mechanism that mediates the return from the 'on' to the 'off'-state. It is usually slow but can be accelerated by orders of magnitude upon inter-action with GTPase-activating proteins (GAPs). In the case of Ras, a major regulator of cellular growth, point mutations are found in approximately 30% of human tumours which render the protein unable to hydrolyse GTP, even in the presence of Ras-GAPs. The first structure determination of a GTPase-activating protein reveals the catalytically active fragment of the Ras-specific p120GAP (ref. 2), GAP-334, as an elongated, exclusively helical protein which appears to represent a novel protein fold. The molecule consists of two domains, one of which contains all the residues conserved among different GAPs for Ras. From the location of conserved residues around a shallow groove in the central domain we can identify the site of interaction with Ras·GTP. This leads to a model for the interaction between Ras and GAP that satisfies numerous biochemical and genetic data on this important regulatory process.

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, γ-tubulin, TUBGCP2, TUBGCP3) and regulatory (HDAC6) microtubule components as detected by co-immunoprecipitation 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.

The sirtuin SIRT4 has been implicated in the control of autophagy and mitochondrial quality control via mitophagy, but the regulatory role of SIRT4 in autophagy/mitophagy induced by different stressors is unclear. Here, we show that cells expressing SIRT4(H161Y), a catalytically inactive, dominant-negative mutant of SIRT4, fail to upregulate LC3B-II and show a reduced autophagic flux upon treatment with different inducers of mitophagy/autophagy, i.e., CoCl2-triggered pseudohypoxia, CCCP/oligomycin-mediated respiratory chain inhibition, or rapamycin treatment. Interestingly, SIRT4(H161Y) expression (i) upregulated protein levels of HDAC6, which is involved in mitochondrial trafficking and autophagosome-lysosome fusion, and (ii) inhibited the conversion of OPA1-L to OPA1-S, which is associated with increased mitochondrial fusion and decreased mitophagy. Both HDAC6 and OPA1 are SIRT4 interactors. However, pharmacological inhibition of neither HDAC6 via Tubacin nor OPA1 via MYLS22 restored the stress-induced upregulation of LC3B-II levels upon autophagy/mitophagy treatment of SIRT4(H161Y)-expressing cells. Remarkably, inhibition of autophagosome-lysosome fusion and thus disruption of late autophagic flux by BafA1 treatment also failed to restore LC3B-II levels upon autophagy/mitophagy treatment, suggesting an inhibitory effect of SIRT4(H161Y) on the initiation/early phase of autophagy. Consistent with this idea, we show that SIRT4(H161Y) promoted the phosphorylation of ULK1 at S638 and S758 (mTORC1 targets), both of which mediate an important inhibitory regulation of autophagy initiation. Thus, our data suggest a positive regulatory function of SIRT4, presumably via modulation of AMPK/mTORC1 signaling, in the ULK1-dependent early regulation/initiation of stress-induced autophagic flux.