Biochimica et Biophysica Acta (BBA) - Molecular Cell Research

It has been proposed that arachidonic acid stimulates neurite outgrowth by specifically activating syntaxin 3, a SNARE proteins that mediates the fusion of transport vesicles with the plasma membrane and is thus instrumental for the insertion of new membrane needed for cell growth and proliferation. Considering the important role of arachidonic acid and other polyunsaturated fatty acids for human health, these findings would have wide implications for everyday life. Here, I have report that the effect of arachidonic acid is caused by a nonspecific, detergent-like action on SNARE proteins in vitro. The action requires unphysiologically high concentrations, is mimicked by several detergents or detergent-like substances forming micelles regardless of whether arachidonic acid is present or not, and is thus highly unlikely to be of any physiological significance for SNARE function.

In previous papers, using the eGFP-RBD3 probe, which binds Ras-GTP with high affinity, we showed that activated Ras proteins are localized to the plasma membrane and in the nucleus in wild-type Saccharomyces cerevisiae cells growing exponentially on glucose, while an aberrant accumulation of activated Ras in mitochondria correlates to mitochondrial dysfunction, accumulation of ROS and an increase of apoptosis. In this paper, we show that lack of TPS1, which is known to trigger apoptosis in S. cerevisiae, induces localization of active Ras proteins in mitochondria, confirming the above-mentioned correlation. Next, by characterizing the ras1{Delta} and ras2{Delta} mutants concerning localization of active Ras proteins and propensity to undergo cell death, we show that active Ras2 proteins, which accumulate in the mitochondria following addition of acetic acid, a well-known pro-apoptotic stimulus, might be the GTPases involved in regulated cell death, while active Ras1 proteins, constitutively localized in mitochondria, might be involved in a pro-survival molecular machinery. Finally, by characterizing the gpa2{Delta} and cyr1{Delta} mutants concerning the propensity to undergo cell death, we show that active mitochondrial Ras proteins promote apoptosis through the cAMP/PKA pathway.

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.

HID1 domain-containing protein 1 (HID1) is a Golgi apparatus (GA) protein involved in the trafficking of cellular material. From reduced expression in cancer cell lines, it had been proposed to be a tumor suppressor in humans. In contrast, it is required for normal larval development of Caenorhabditis elegans, thereby raising an apparent contradiction for HID1 function regarding the inhibition or maintenance of cell proliferation. An extensive comparison of publicly available gene expression data revealed that HID1 transcript levels in cancer cell lines were not representative of those in primary tumors, and the gene is amplified in many primary tumors. These findings question the role for its expression to suppress cell proliferation. Subsequently, we employed the model S. pombe to explore the role of HID1 in cell proliferation. The knock-out mutant hid1{Delta} exhibited a reduced proliferation phenotype due to a prolonged lag-phase but, eventually, attained parent strain rates of apparent proliferation. RNAseq-based transcriptomics revealed that hid1{Delta} retained features of metabolically stressed cells with quiescence-like transcriptional regulation. A label-free proteomic analysis revealed a lack of various membrane proteins suggesting cells with compromised Golgi apparatus (GA) function. The inability of hid1{Delta} to grow on agar containing a standard minimal medium suggested a link to metabolic deficiency. These findings point to a hid1{Delta}-related perturbation in GA function that compromises cell recovery from metabolic change.

Monomers, dimers, and individual FOF1-ATP synthase subunits are, presumably, involved in the formation of the mitochondrial permeability transition pore (PTP), which molecular structure, however, is still unknown. We hypothesized that upon the Ca2+-dependent assembly of PTP complex, F-ATP synthase (subunits) recruits mitochondrial proteins that do not interact or weakly interact with F-ATP synthase under normal conditions. Therefore, we examined whether the PTP opening in mitochondria before the separation of supercomplexes by BN-PAGE will increases the channel stability and channel-forming capacity of isolated F-ATP synthase dimers and monomers in planar lipid membranes. Besides, we studied the specific activity and protein composition of F-ATP synthase dimers and monomers from rat liver and heart mitochondria before and after PTP opening. By contrast to our expectations, preliminary PTP opening dramatically suppressed the high-conductance channel activity of F-ATP synthase dimers and monomers and decreased their specific "in gel" activity. The decline in the channel-forming activity correlated with the reduced levels of as few as two proteins in the bands: methylmalonate-semialdehyde dehydrogenase and prohibitin 2. These data indicate that proteins accompanying F-ATP synthase may be important players in the PTP formation and stabilization.

Mitochondria dysfunction is involved in the pathomechanism of many illnesses including Parkinsons disease. PINK1, which is mutated in some cases of familiar Parkinsonism, is a key component in the degradation of damaged mitochondria by mitophagy. The accumulation of PINK1 on the mitochondrial outer membrane (MOM) of compromised organelles is crucial for the induction of mitophagy, but the molecular mechanism of this process is still unresolved. Here, we investigate the association of PINK1 with the TOM complex. We demonstrate that PINK1 heavily relies on the import receptor TOM70 for its association with mitochondria and directly interacts with this receptor. The structural protein TOM7 appears to play only a moderate role in PINK1 association with the TOM complex, probably due to its role in stabilizing this complex. PINK1 requires the TOM40 pore lumen for its stable interaction with the TOM complex and apparently remains there during its further association with the MOM. Overall, this study provides new insights on the role of the individual TOM subunits in the association of PINK1 with the MOM of depolarized mitochondria.

Phosphatidic acid (PA) and Lysophosphatidic acid acyltransferases (LPAATs) might be critical for the secretory pathway. Four extra-plastidial LPAATs (numbered 2,3,4 and 5) were identified in A. thaliana. These AtLPAATs, displaying an enzymatic activity specific for LPA to produce PA, are located in the endomembrane system. We focused on the putative role of the AtLPAATs 3, 4 and 5 in the secretory pathway of root cells through genetical (knock-out mutants), biochemical (activity inhibitor, lipid analyses) and imaging (live and immuno-confocal microscopies) approaches. Treating a lpaat4;lpaat5 double mutant with the LPAAT inhibitor CI976 showed a primary root growth decrease. The transport of the auxin transporter PIN2 was disturbed in this lpaat4;lpaat5 double mutant treated with CI976, but not that of H+-ATPases. The lpaat4;lpaat5 double mutant was sensitive to salt stress and the transport of the aquaporin PIP2;7 to the plasma membrane in the lpaat4;lpaat5 double mutant treated with CI976 was reduced. We measured the amounts of neo-synthesized PA in roots, and found a decrease in PA only in the lpaat4;lpaat5 double mutant treated with CI976, suggesting that the protein transport impairment was due to a critical PA concentration threshold. HighlightPhosphatidic acid produced by Lyso-Phosphatidic Acid Acyl-Transferases has an impact on the efficiency of the intracellular transport of some proteins in Arabidopsis thaliana root cells.

Phospholipases D1 and D2 (PLD1/2) have been implicated in tumorigenesis. We previously detected higher expression of PLD in the nuclei of patient-derived prostate cancer (PCa) cells and prostate cancer cell lines. Here we have examined whether PLD1 or PLD2 are associated with the nuclear matrix and influence cell cycling. PLD1/PLD2 were detected by qualitative immunofluorescence in cultured PCa cells and extracted with a standardised protocol to reveal nuclear matrix-associated proteins. The effects of isoform-specific inhibition of PLD1or PLD2 on PCa cell cycle progression were analysed by flow cytometry. PLD2 mainly co-localised with the nucleolar marker fibrillarin in PCa cells. However, even after complete extraction, some PLD2 remained associated with the nuclear matrix. Inhibiting PLD2 effectively reduced PCa cell cycling into and through S phase. In contrast, PLD1 inhibition effects were weaker, and a subpopulation of cycling patient-derived PCa cells was unaffected by PLD1 inhibition. When associated with the nuclear matrix PLD2 could generate phosphatidic acid to regulate nuclear mTOR and control downstream transcriptional events. The association of PLD2 with the nucleolus also implies a role in stress regulation. The cell cycling results highlight the importance of PLD2 inhibition as a novel potential prostate cancer therapeutic mechanism by differential regulation of cell proliferation.

Ovarian cancer stands out as one of the tumors with a high mortality rate in women. Therefore, the search for new therapeutic targets is essential to enhance patient prognosis. There is evidence suggesting that Hook2, an adaptor protein involved in microtubule transport, may play a role in cancer, particularly ovarian cancer. This study examines the role of HOOK2 in the progression of ovarian tumors. The findings reveal that a decrease in HOOK2 levels leads to diminished growth and cellular migration in ovarian cancer cells, impeding the in vivo formation of tumors. The reduction of HOOK2 is associated with both an increase in endoplasmic reticulum stress and an elevation in cell death, the latter likely caused by the activation of the unfolded protein response. Moreover, the study observes that the decrease in HOOK2 diminishes the properties of cancer stem cells in ovarian cancer, possibly due to the increase in cell death specifically found within these stem cells. Given the profound impact of reduced HOOK2 levels on ovarian cancer cells, this gene emerges as a promising therapeutic strategy for treating ovarian cancer patients.

The small GTPase Rho5 acts as a central hub to mediate the yeasts response to adverse environmental conditions, including oxidative stress, with the concomitant induction of mitophagy and apoptosis. A proper cellular stress response has been correlated with the rapid translocation of the GTPase to the mitochondria, which depends on its activating dimeric GDP/GTP exchange factor (GEF). Here, the small ALFA tag was attached to Rho5 or the GEF subunits Dck1 and Lmo1 to efficiently trap the functional fusion proteins to specific cellular membranes, i.e. the plasma membrane, the mitochon-drial outer membrane, or the nuclear membrane, via fusions of integral membrane proteins residing in these compartments with an ALFA nanobody. The trapped components were subjected to life-cell fluorescence microscopy in combination with GFP fusions of the GTPase or its GEF subunits to investigate their interaction in vivo. We found that the dimeric GEF tends to auto-assemble and form stable dimers independent of its intracellular localization. On the other hand, GFP-Rho5 does not stably colocalize with the trapped GEF, attributed to its transient interaction. Phenotypic analyses of strains with the misslocalized proteins indicate that for a proper oxidative stress response Lmo1 needs to associate with the plasma membrane. In contrast, Rho5 only exerts its role at the mitochondrial surface when it is there in its active conformation. These data underline the importance of the proper spatio-temporal distribution of Rho5-GTP during oxidative stress response.

Dietary methionine restriction is associated with a reduction in tumor growth in preclinical studies and an increase in lifespan in animal models. The mechanism by which methionine restriction inhibits tumor growth while sparing normal cells is incompletely understood, except for the observation that normal cells can utilize methionine or homocysteine interchangeably (methionine independence) while most cancer cells are strictly dependent on methionine availability. Here, we compared a typical methionine dependent and a rare methionine independent melanoma cell line. We found that replacing methionine with homocysteine generally induced hypomethylation in gene promoters. We isolated nuclear proteins and submitted it for tandem mass tag (TMT) proteomics. This analysis revealed that several proteins involved in the mitochondrial integrated stress response (ISR) were upregulated in response to the replacement of methionine to homocysteine in both cell lines, but to a much greater degree in the methionine dependent cell line. Consistent with the ISR signature, a proteomic analysis of a subcellular fraction enriched for mitochondrial content revealed a strong enrichment for proteins involved in oxidative phosphorylation. Analysis of cellular bioenergetics confirmed that homocysteine induces a decrease in ATP production from oxidative phosphorylation and glycolysis, but to a similar extent in methionine dependent and methionine independent cells. The mitochondrial integrated stress response shared a signature with ferroptosis. Methionine dependent cells displayed a strong ferroptotic signature, which was decreased by half in methionine independent cells. Consistent with ferroptosis, lipid peroxidation was significantly increased in methionine independent cells grown in homocysteine, and viability could be rescued partially but significantly with the inhibitor ferrostatin. Therefore, we propose that methionine stress induces ferroptotic cell death in methionine dependent cancer cells.

Treatment of endothelial cells with bacterial lipopolysaccharide (LPS) evokes a number of metabolic and functional consequences which built a multifaceted physiological response of endothelium to bacterial infection. Here effects of LPS on human aortic endothelial cells (HAEC) have been investigated. Among the spectrum of biochemical changes substantially elevated N-nicotinamide methyltransferase (NNMT) protein level was particularly intriguing. This important enzyme may potentially affect cellular metabolism by two means: direct regulation of methylnicotinamide level and availability of nicotinamide, that at least potentially may influence NAD+ synthesis, and regulation of S-adenosylmethionine concentration and therefore controlling methylation of many proteins including chromatin. This may have epigenetic consequences. This paper is focused on NNMT, despite the fact that in the presence of LPS additional effects of this compound mask pure (canonical) consequences of the elevated NNMT protein which are an increased MNA synthesis or reduced NAD+ level. On the other hand, however, it has been shown that silencing of the NNMT-encoding gene prevents several changes which are observed in control HAECs treated with LPS. They include significantly increased calcium response to thapsigargin (store-operated calcium entry), altered energy metabolism which is switched to anaerobic glycolysis and rearrangement of the mitochondrial network. However, a biochemical mechanism behind the protective consequences of the NNMT deficiency in cells treated with LPS remains unexplained.

Nuclear pores control nucleo-cytoplasmic trafficking and directly or indirectly regulate vital cellular processes. Nup88, important for Crm1 mediated nuclear export process, is overexpressed in many cancers. A positive correlation exists between progressive stages of cancer and Nup88 expression. However, links between Nup88 overexpression and head and neck cancer are insignificant, and mechanistic details are non-existent. Here, we report that Nup88 exhibits positive correlation in head and neck cancer in addition to elevated Nup62 levels. We demonstrate that Nup88 interacts with Nup62 in a cell-cycle and glycosylation independent manner. The overexpression of Nup88 or Nup62 imparts proliferation and migration advantages to cells. We further report that the interaction with Nup62 stabilizes Nup88 by inhibiting proteasome-mediated degradation of overexpressed Nup88. Overexpressed Nup88 is stabile and partly inside the nucleus and can interact with NF{kappa}B (p65). Nup88 overexpression induces proliferative and inflammatory responses downstream of p65. Altogether, we suggest that simultaneous overexpression of Nup62 and Nup88 in head and neck cancer stabilizes overexpressed Nup88. Stable Nup88 interacts with p65 and induces inflammatory, proliferative, and migratory advantages to cells, which perhaps is the underlying mechanism driving tumorigenic transformations.

Gap junctions formed by the major neuronal connexin Cx36 function as electrical synapses in the nervous system and provide unique functions such as synchronizing activities or network oscillations. Although the physiological significance of electrical synapses for neuronal networks is well established, little is known about the pathways that regulate the transport of its main component: Cx36. Here we have used HEK293T cells as an expression system in combination with siRNA and BioID screens to study the transition of Cx36 from the ER to the cis Golgi. Our data indicate that the C-terminal tip of Cx36 is a key factor in this process, mediating binding interactions with two distinct components in the early secretory pathway: the COPII complex and the Golgi stacking protein Grasp55. The C-terminal amino acid valine serves as an ER export signal to recruit COPII cargo receptors Sec24A/B/C at ER exit sites, whereas the PDZ binding motif "SAYV" mediates an interaction with Grasp55. These two interactions have opposing effects in their respective compartments. While Sec24 subunits carry Cx36 out of the ER, Grasp55 stabilizes Cx36 in the Golgi as shown in over expression experiments. These early regulatory steps of Cx36 are expected to be essential for the formation, function, regulation and plasticity of electrical synapses in the developing and mature nervous system.

Peroxisomes are membrane-enclosed organelles with important roles in fatty acid breakdown, glycolysis, and biosynthesis of sterols and ether lipids. Defects in peroxisome biogenesis result in severe neurological diseases, such as Zellweger syndrome, neonatal adrenoleukodystrophy, infantile Refsum disease, and myelopathies. However, many aspects of peroxisomal biogenesis are not well understood. Here we investigated delivery of tail-anchored (TA) proteins to peroxisomes in mammalian cells. Using glycosylation assays we showed that peroxisomal TA proteins do not enter ER in both WT and peroxisome-lacking cells. We observed that in cells lacking the essential peroxisome biogenesis factor, PEX19, peroxisomal TA proteins localize mainly to mitochondria. However, in PEX3 KO cells, which lack peroxisomes as well, the endogenous TA protein, ACBD5, does not target mitochondria, suggesting that PEX3 plays an important role in targeting of peroxisomal TA proteins to mitochondria. Finally, to investigate peroxisomal TA protein targeting in cells with fully functional peroxisomes we used a proximity biotinylation approach. We showed that while ER-targeted TA construct was exclusively inserted into the endoplasmic reticulum (ER), peroxisome-targeted TA construct was inserted to both peroxisomes and mitochondria. Thus, in contrast to previous studies, our data suggest that peroxisomal TA proteins do not insert to the ER prior to their delivery to peroxisomes. Instead, mitochondria can play a role in the targeting of TA proteins to peroxisomes.

The biogenesis of small extracellular vesicles (sEVs) is only partially understood. Our recent findings provide evidence that a newly described sEV secretion pathway, the amphiectosome release and the "torn bag mechanism", is present in all tested cell lines and in mouse liver and kidney. Surprisingly, in in situ fixed steady-state cells, transmission electron microscopy did not reveal the classical exosome secretion route, the sEV release via exocytosis of multivesicular endosomes (MVEs). In the current study, we investigated which parameters influence the activation of the two distinct sEV release mechanisms. Our results show that under stress conditions (such as Ca{superscript 2} ionophore-induced membrane stress or metabolic stress-induced by serum starvation), exocytosis of MVEs is activated, while this process is absent in steady-state conditions. By silencing ATG5 (a key regulator of autophagy) and RAB27a (essential small GTPase for MVE exocytosis), we selectively modulated these two mechanisms. Amphiectosome release depended on both autophagy and ATG5, while exocytosis of MVE was autophagy-independent but RAB27a-dependent. Our findings suggest that sEV release via the "torn bag mechanism" is a general and essential secretion pathway in non-stressed, steady-state mammalian cells, while stress conditions induce the sEV release via MVE exocytosis.

Glycerophospholipid (GPL) homeostasis in eukaryotic cells is thought to be maintained via biosynthesis, degradation and acyl chain remodeling. Here we provide evidence for an additional process termed "head-group remodeling" where other GPLs, when in excess, are rapidly converted to phosphatidylcholine and triacylglycerol. Mass spectrometric studies showed the formation of diacylglycerol, but not phosphatidic acid, from the exogenous GPL thus indicating that the first step is catalyzed by a phospholipase C-type enzyme. Consistently, triacylglycerol formation was significantly inhibited by the knock-down of several PLCs, but not phospholipase Ds. Second, we found that each exogenous GPL strongly inhibited the synthesis of the corresponding endogenous GPL class. Based on these and previous data we hypothesize how mammalian cells could coordinate the multiple processes contributing to GPL homeostasis in mammalian cells. In conclusion, this study provides the first evidence that head group remodeling plays an important role in GPL homeostasis in mammalian cells.

NF-{kappa}B is primarily known for its transcriptional activation function in the context of immune responses, inflammation, cell survival, proliferation and Cancer. However, whether NF-{kappa}B functions as a transcriptional repressor in the context of tumor growth has not been well addressed. While, classical NF-kB activated by IKK-b has been shown to play a tumor promoting role in colorectal cancer, couple of studies suggested that overexpression of RelA in colorectal cancer cells leads to apoptosis. These findings were contradictor and puzzling with regards to the role of RelA in colorectal cancer. Here, we report that RelA represses proapoptotic gene puma and contributes to colorectal cancer cell survival. Interestingly, Yin-Yang1, a RelA target gene product is also essential to repress puma and contributes to colorectal cancer cell survival. Moreover, RelA and YY1 form a complex in colorectal cancer cells. Depletion of either RelA or YY1 results in upregulation of pro-apoptotic gene Puma. Importantly, we show that binding of Yin Yang1 to RelA impairs transcriptional activation by RelA suggesting that YY1 is an inhibitor of RelA function. Collectively, we present evidence for RelA-YY1 complex formation in colorectal cancer cells, both RelA and YY1 function as transcriptional repressors of puma and contribute to survival of colorectal cancer cells.

ATAD1 is an AAA-ATPase which shows a dual localization at mitochondria and peroxisomes. While its peroxisomal function is not known, in mitochondria ATAD1 is part of a quality control mechanism extracting mislocalised tail-anchored and accumulated precursor proteins from the outer membrane. Here, we studied the peroxisomal interactome of ATAD1 and could show that human ATAD1 interacts with PEX5, a cytosolic receptor for peroxisomal matrix proteins which transiently inserts into peroxisomal membranes. Upon cargo-release, mono-ubiquitinated PEX5 is recycled into the cytosol by the AAA-peroxins PEX1 and PEX6. The accumulation of ubiquitinated PEX5 is known to trigger degradation of whole organelles called pexophagy. Here, we used ATAD1-, PEX1- and ATAD1/PEX1-CRISPR-Knockout cell lines to investigate the physiological role of an ATAD1-PEX5 interaction. We could show an influence of ATAD1 on the stability of accumulated PEX5 and hypothesize a role in a peroxisomal quality control mechanism enabling clearance of ubiquitinated receptor from the membrane.

Colorectal cancer is the second leading cause of cancer-related deaths worldwide, highlighting the need for improved treatments and advanced molecular research. A recent therapeutic approach focuses on repurposing drugs to target dysregulated pathways involved in tumorigenesis. Among these, statins, commonly known for lowering cholesterol, have attracted attention for their potential anti-cancer properties. Here, we provide direct evidence for the same by assessing the impact of statin treatment on lipid, transcript, and protein levels. Our findings reveal that statins specifically target key components of the Wnt/{beta}-catenin pathway, a major factor in adenoma formation, including the SATB (Special AT-rich Binding protein) family proteins. While SATB1 is recognized as a regulator of tumorigenesis, particularly under Wnt signaling, SATB2 appears to exert an opposing role. We demonstrate that statin treatment reciprocally alters the expression pattern of these proteins. Furthermore, a human clinical trial evaluating statins as an anti-cancer therapy supports the hypothesis that differential expression of SATB proteins is crucial in tumorigenic outcomes. In conclusion, this modulation by statin treatment suggests promising new therapeutic avenues through drug repurposing.