Biology subjects

Ahmadian, M. R.

Publications and source records attributed to Ahmadian, M. R..

7 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↗

bioRxiv2025

New insights into the classification of the RAC1 P29S hotspot mutation in melanoma as an oncogene

The RAC1P29S hotspot mutation, prevalent in melanoma, drives tumorigenesis by enhancing molecular interactions and hyperactivating key signaling pathways, making it a compelling target for cancer therapy. This study provides a comprehensive biochemical characterization of RAC1P29S compared to wild-type RAC1 and mutations T17N and F28L. The P29S mutation significantly impairs nucleotide binding to guanosine triphosphate (GTP) and guanosine diphosphate, accelerating intrinsic nucleotide exchange. While minimally affecting regulation by guanosine dissociation inhibitor 1, RAC1P29S exhibits reduced activation via diffuse B-cell lymphoma family guanine nucleotide exchange factors but retains effective activation by dedicator of cytokinesis 2. Critically, the P29S mutation severely impairs GTPase-activating protein-stimulated GTP hydrolysis, most likely contributing to RAC1P29S hyperactivation by prolonging its GTP-bound form. RAC1P29S displays a stronger binding affinity for IQ motif-containing GTPase-activating protein 1 than for p21-activated kinase 1, highlighting the role of the former in scaffolding RAC1P29S-driven signaling. In serum-starved cells, RAC1P29S predominantly adopts an active GTP-bound state. RAC1P29S overexpression activates key cancer-associated pathways, including extracellular signal-regulated kinase and p38 mitogen-activated protein kinase, reinforcing its role as an oncogenic driver in melanoma. These insights suggest potential therapeutic targets for melanoma treatment, including RAC1 regulators and modulators. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=93 SRC="FIGDIR/small/637030v1_ufig1.gif" ALT="Figure 1"> View larger version (41K): org.highwire.dtl.DTLVardef@2385c2org.highwire.dtl.DTLVardef@229a31org.highwire.dtl.DTLVardef@1b93011org.highwire.dtl.DTLVardef@102b427_HPS_FORMAT_FIGEXP M_FIG C_FIG A model of RAC1P29S activation and signaling in cancer cells. RAC1P29S remains in an inactive GDP-bound state in the cytoplasm where GDI1 prevents its membrane association. Upon stimulation, GEFs, primarily DOCK2, activate RAC1P29S by promoting GDP-GTP exchange, facilitating its transition to the active GTP-bound state and initiating downstream signaling. RAC1P29S binds preferentially to IQGAP1 over PAK1, reflecting a shift in effector interactions. IQGAP1 acts as a scaffolding protein, spatially modulating RAC1P29S-driven signaling and amplifying its effects. Under normal conditions, GAPs such as p50GAP regulate RAC1 by accelerating GTP hydrolysis, thereby maintaining its dynamic activation cycle. However, the P29S mutation severely impairs p50GAP-mediated hydrolysis, leading to accumulation of RAC1P29S in its GTP-bound state and loss of temporal regulation. This persistent activation hyperactivates downstream effectors and promotes cancer-associated pathways, including ERK and p38 MAPK, which drive cell growth, survival, invasion and metastasis.

cancer biology↗

bioRxiv2023

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

Cellular responses leading to development, proliferation, and differentiation rely on RAF/MEK/ERK signaling that integrates and amplifies signals from various stimuli to cellular downstream responses. The clinical significance of C-RAF activation has been reported in many types of tumor cell proliferation and developmental disorders, which requires the discovery of potential C-RAF protein regulators. Here, we identify a novel and specific protein interaction between C-RAF, among the RAF kinase paralogs, and SIRT4 among the mitochondrial sirtuin family members SIRT3, SIRT4, and SIRT5. Structurally, C-RAF binds to SIRT4 through the N-terminal cysteine-rich domain (CRD; a.a. 136-187), and on the other side, SIRT4 requires predominantly the C-terminus (a.a. 255-314) for full interaction with C-RAF. Interestingly, SIRT4 interacts specifically with C-RAF in a pre-signaling inactive (serine 259 phosphorylated) state. Consistent with this finding, ectopic expression of SIRT4 in HEK293 cells results in upregulation of pS259-C-RAF levels and concomitant reduction of MAPK signaling as evidenced by strongly decreased phospho-ERK signals. Thus, our findings propose another extra-mitochondrial role of SIRT4 and suggest that SIRT4 functions as a cytosolic tumor suppressor of C-RAF-MAPK signaling, besides its known metabolic tumor suppressor role towards glutamate dehydrogenase and glutamine levels in mitochondria.

biochemistry↗

bioRxiv2023

CoCl2 triggered pseudohypoxic stress induces proteasomal degradation of SIRT4 via polyubiquitination of lysines K78 and K299

SIRT4 comprises together with SIRT3 and SIRT5 the mitochondrially localized subgroup of sirtuins. SIRT4 regulates via its NAD+-dependent enzymatic activities mitochondrial bioenergetics, dynamics (mitochondrial fusion), and quality control (mitophagy). Here, we address the regulation of SIRT4 itself by characterizing its protein stability and degradation upon CoCl2-induced pseudohypoxic stress that typically triggers mitophagy. Interestingly, within the mitochondrial sirtuins, only the protein levels of SIRT4 or ectopically expressed SIRT4-eGFP decrease upon CoCl2 treatment of HEK293 cells. Co-treatment with BafA1, an inhibitor of autophagosome-lysosome fusion required for autophagy/mitophagy, or the use of the proteasome inhibitor MG132 prevented CoCl2-induced SIRT4 downregulation. Consistent with the proteasomal degradation of SIRT4, the lysine mutants SIRT4(K78R) and SIRT4(K299R) showed significantly reduced polyubiquitination upon CoCl2 treatment and were more resistant to pseudohypoxia-induced degradation as compared to SIRT4. Moreover, SIRT4(K78R) and SIRT4(K299R) displayed increased basal protein stability as compared to wild-type SIRT4 when subjected to MG132 treatment or cycloheximide (CHX) chase assays. Thus, our data indicate that stress-induced protein degradation of SIRT4 occurs through two mechanisms, (i) via mitochondrial autophagy/mitophagy, and (ii) as a separate process via proteasomal degradation within the cytoplasm.

biochemistry↗

bioRxiv2023

Characterization of the small Arabidopsis thaliana GTPase and ADP-ribosylation factor-like 2 protein TITAN5

Small GTPases function by conformational switching ability between GDP- and GTP-bound states in rapid cell signaling events. The ADP-ribosylation factor (ARF) family is involved in vesicle trafficking. Though evolutionarily well conserved, little is known about ARF and ARF-like GTPases in plants. Here, we characterized biochemical properties and cellular localization of the essential small ARF-like GTPase TITAN 5/HALLIMASCH/ARL2/ARLC1 (hereafter termed TTN5) from Arabidopsis thaliana. Two TTN5 variants were included in the study with point mutations at conserved residues, suspected to be functional for nucleotide exchange and GTP hydrolysis, TTN5T30N and TTN5Q70L. We found that TTN5 had a very rapid intrinsic nucleotide exchange capacity with a conserved nucleotide switching mechanism. TTN5 acted as a non-classical small GTPase with a remarkably low GTP hydrolysis activity, suggesting it is likely present in GTP-loaded active form in the cell. We analyzed signals from yellow fluorescent protein (YFP)-tagged TTN5 and from in situ immunolocalization of hemagglutine-tagged HA3-TTN5 in Arabidopsis seedlings and in a transient expression system. Together with colocalization using endomembrane markers and pharmacological treatments the microscopic analysis suggests that TTN5 can be present at the plasma membrane and dynamically associated with membranes of vesicles, Golgi stacks and multivesicular bodies. While the TTN5Q70L variant showed similar GTPase activities and localization behavior as wild-type TTN5, the TTN5T30N mutant differed in some aspects. Hence, the unusual capacity of rapid nucleotide exchange activity of TTN5 is linked with cell membrane dynamics, likely associated with vesicle transport pathways in the endomembrane system. HighlightsO_LIThe small ARF-like GTPase TTN5 has a very rapid intrinsic nucleotide exchange capacity with a conserved nucleotide switching mechanism C_LIO_LIBiochemical data classified TTN5 as a non-classical small GTPase, likely present in GTP-loaded active form in the cell C_LIO_LIYFP-TTN5 is dynamically associated with vesicle transport and different processes of the endomembrane system, requiring the active form of TTN5 C_LI

plant biology↗

bioRxiv2022

Alteration of myocardial structure and function in RAF1associated Noonan syndrome: Insights from cardiac disease modeling based on patient-derived iPSCs

Noonan syndrome (NS), the most common among the RASopathies, is caused by germline variants in genes encoding components of the RAS-MAPK pathway. Distinct variants, including the recurrent Ser257Leu substitution in RAF1, are associated with severe hypertrophic cardiomyopathy (HCM). Here, we investigated the elusive mechanistic link between NS-associated RAF1S257L and HCM using three-dimensional cardiac bodies and bioartificial cardiac tissues generated from patient-derived induced pluripotent stem cells (iPSCs) harboring the pathogenic RAF1 c.770C>T missense change. We characterize the molecular, structural and functional consequences of aberrant RAF1 -associated signaling on the cardiac models. Ultrastructural assessment of the sarcomere revealed a shortening of the I-bands along the Z disc area in both iPSC-derived RAF1S257L cardiomyocytes, and myocardial tissue biopsies. The disease phenotype was partly reverted by using both MEK inhibition, and a gene-corrected isogenic RAF1L257S cell line. Collectively, our findings uncovered a direct link between a RASopathy gene variant and the abnormal sarcomere structure resulting in a cardiac dysfunction that remarkably recapitulates the human disease. These insights represent a basis to develop future targeted therapeutic approaches.

cell biology↗

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↗