SIRT4 positively regulates autophagy via ULK1, but independently of HDAC6 and OPA1

Lehmkuhl, I.; Amin, K.; Hampel, N.; Iram, A.; Hesse, J.; Wiek, C.; Nguyen, J. T. V.; Hanenberg, H.; Scheller, J.; Ahmadian, M. R.; Stork, B.; Floss, D. M.; Piekorz, R. P.

doi:10.1101/2025.04.29.649368

bioRxiv · 10.1101/2025.04.29.649368

SIRT4 positively regulates autophagy via ULK1, but independently of HDAC6 and OPA1

Lehmkuhl, I.·Amin, K.·Hampel, N.·Iram, A.·Hesse, J.·Wiek, C.·Nguyen, J. T. V.·Hanenberg, H.·Scheller, J.·Ahmadian, M. R.·Stork, B.·Floss, D. M.·Piekorz, R. P.

View original source ↗Export metadata ↓

Abstract

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.

Source connections

Published article ↗

Explore related subjects

biochemistry Preprint

Keep this discovery

Explore connections, maps & timelines

BibTeX RIS

Lehmkuhl, I., Amin, K., Hampel, N., Iram, A., Hesse, J., Wiek, C., Nguyen, J. T. V., Hanenberg, H., Scheller, J., Ahmadian, M. R., Stork, B., Floss, D. M., Piekorz, R. P.. 2025-04-29. SIRT4 positively regulates autophagy via ULK1, but independently of HDAC6 and OPA1. https://doi.org/10.1101/2025.04.29.649368

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related preprints

bioRxiv2026

MgATP/MgADP-dependent conformational dynamics and intrinsically disordered regions of vascular KATP channels revealed by cryoEM

Vascular smooth muscle KATP channels, composed of the pore-forming Kir6.1 and regulatory SUR2B subunits, control vascular tone, dysfunction of which causes systemic disease. Vascular KATP is regulated by Mg-nucleotides, but the underlying structural mechanism has remained elusive. Here, we determined cryoEM structures of these channels in the presence of MgATP and MgADP. Two key structures captured, one showing the SUR2B-nucleotide binding domains (NBDs) separated and one showing the SUR2B-NBDs dimerized, reveal conformation-specific organization of intrinsically disordered regions (IDRs) found in both Kir6.1 and SUR2B. In the NBD-separated conformation, the Kir6.1-N terminal IDR (KNt) sits within the central cleft of the ABC-core of SUR2B. In the NBD-dimerized conformation, KNt is excluded from the central cleft and instead forms contacts with an ED domain comprising 15 consecutive glutamate and aspartate residues within a SUR2B IDR, the N1-T2 linker connecting NBD1 (N1) to transmembrane domain 2 (T2). Moreover, within the N1-T2 linker a regulatory helix seen between the two NBDs in the NBD-separated conformation moves to outside the dimerized NBDs, interacting with the C-terminal residues unique to SUR2B, in the NBD-dimerized conformation. MD simulations further reveal that transient but frequent interactions mediated by the IDRs may facilitate Mg-nucleotide dependent conformational switch in vascular KATP channels.

biochemistry↗

bioRxiv2026

Probing the sequence variability tolerance in a de novo α-helical barrel biocatalyst

De novo-designed enzymes have recently achieved high catalytic activity and stereoselectivity while demonstrating exceptional thermostability in entirely novel protein scaffolds. Among these, -helical barrel protein scaffolds are attractive structures for biocatalysis due to their structural simplicity, high thermostability, and rationalizable sequence patterning. However, enabling major structural reengineering of these scaffolds while maintaining the structure, stability and catalytic activity while also improving soluble protein production remain major challenges and pose the fundamental question how engineerable a de novo backbone-sequence pair is. Here, we combine deep learning based and classic computational protein design to modify and optimize de novo -helical barrel biocatalysts. Using the previously reported six-helical barrel 6H5L as a model scaffold, AlphaFold2-guided RosettaRemodel enabled the design of a truncated variant, whose crystal structure closely matches the design model. Additional sequence-redesign using ProteinMPNN generated a variant with a tenfold increase of soluble protein yield in Escherichia coli. Biochemical, biophysical, and structural analyses showed that both variants retained the overall barrel architecture, high thermal stability, and catalytic activity for both purified protein and whole-cell systems. Detailed kinetic analysis on the variants showed both variation in kcat and Km, reflecting changes in catalytic turnover and substrate binding. Together, these approaches provide new insights and possibilities for the further engineering of functional de novo -helical barrels, their ability to withstand dramatically large sequence changes and their broader application in biocatalysis and biotechnology.

biochemistry↗

bioRxiv2026

Cytokine-induced nuclear translocation of STAT1 via a non-transferable NLS

The targeting function of nuclear localization signals (NLSs) is generally considered independent of a protein's native sequence or fold and is readily transferable to heterologous cargos. Contrary to this paradigm, rapid nuclear translocation of phosphorylated STAT1 (pSTAT1) following cytokine stimulation requires importin {beta}, Ran-GTP, and the importin 5 isoform, which recognizes a non-transferable NLS. Here, we present cryo-EM structures of pSTAT1 bound to importin 5, revealing an asymmetric 2:1 complex that diverges from canonical NLS-mediated cargo recognition. Importin 5 occupies the DNA-binding groove of the pSTAT1 dimer, with a single STAT1 N-terminal domain positioning the C-terminal Armadillo repeats 9-10 (S1B domain) orthogonal to the DNA-binding interface. This interface is also targeted by the Ebola virus protein VP24, an antagonist of interferon signaling. We further show that Ran-GTP alone is insufficient to trigger nuclear release of pSTAT1, which additionally requires the exportin CAS. A cryo-EM reconstruction of the CAS-Ran-GTP-5 complex, supported by in vitro competition assays, demonstrates that CAS and pSTAT1 are mutually exclusive ligands for importin 5. Together, these findings define the molecular choreography of cytokine-induced STAT1 nuclear translocation and release, establishing a general paradigm for STAT family signaling.

biochemistry↗