Biology subjects

Wiek, C.

Publications and source records attributed to Wiek, C..

4 recordsLinked to original sources

bioRxiv2025

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

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.

biochemistry↗

bioRxiv2022

A Minimal Model of CD95 Signal Initiation Revealed by Advanced Super-resolution and Multiparametric Fluorescence Microscopy

Unraveling the spatiotemporal organization and dynamical interactions of receptors in the plasma membrane remains a key challenge for our mechanistic understanding of cell signal initiation. A paradigm of such process is the oligomerization of TNF receptor CD95 during apoptosis signaling, where molecular configurations are yet to be defined. Here, we scrutinize proposed oligomerization models in live cells, establishing a molecular sensitive imaging toolkit including time-resolved FRET spectroscopy, quantitative STED microscopy, confocal Photobleaching Step Analysis and FCS. CD95 interactions were probed over molecular concentrations of few to [~] 1000 molecules/{micro}m2, over ns to hours, and molecular to cellular scales. We further established high-fidelity monomer and dimer controls for quantitative benchmarking. Efficient apoptosis was already observed when [~] 8 to 17% monomeric CD95 oligomerize to dimers/trimers after ligand binding. Our multiscale study highlights the importance of molecular concentrations, of the native environment, and reveals a minimal oligomerization model of CD95 signal initiation.

biophysics↗

bioRxiv2020

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

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 (,{beta}-tubulin, {gamma}-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 -tubulin (K40) associated with altered microtubule dynamics in mitotic cells. SIRT4 or the N-terminally truncated variant SIRT4({Delta}N28), 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.

cell biology↗

bioRxiv2019

Ammonia inhibits energy metabolism in astrocytes in a rapid and glutamate dehydrogenase 2-dependent manner

In hepatic encephalopathy (HE) astrocyte dysfunction is a primary factor impairing neuronal activity under hyperammonemia. We show that mitochondria in cellular HE models undergo rapid fragmentation under hyperammonemia in a reversible manner. Mitochondrial respiration and glycolysis were instantaneously hampered in a pH-independent manner. A metabolomics approach revealed a subsequent accumulation of numerous amino acids, including branched chain amino acids, and glucose. N15 labeling of ammonia shows rapid incorporation of ammonia-derived nitrogen into glutamate and glutamate-derived amino acids. Downregulating human GLUD2, encoding mitochondrial glutamate dehydrogenase 2 (GDH2), inhibiting GDH2 activity by SIRT4 overexpression, and supplementing cells with glutamate or glutamine alleviated ammonia-induced inhibition of mitochondrial respiration. Thus, under hyperammonemic conditions, GDH2 catalyzes the removal of ammonia by reductive amination of -ketoglutarate but at the same time inhibits the TCA-cycle by depleting -ketoglutarate. Overall, we propose a mitochondria-dependent mechanism contributing to the early steps in the pathogenesis of HE where the interplay between energy metabolism and ammonia removal plays a pivotal role.

biochemistry↗