Biochimica et Biophysica Acta (BBA) - Molecular Cell Research

Ubp3 is one of about 20 deubiquitinases (DUBs) in S. cerevisiae. The current view generally assumes that Ubp3 requires its interaction partner Bre5, which is proposed to function as a positive regulator. Accordingly, the Ubp3/Bre5 complex has been implicated in a broad range of cellular processes for example trafficking between ER and Golgi, stress granule formation and selective autophagy. However, the molecular basis of this proposed Bre5-dependent activity remains unclear. To address this at a molecular level, Ubp3, Bre5, and related constructs were heterologously expressed in E. coli, purified to homogeneity, and characterized in vitro. Both proteins contain folded domains as well as extensive intrinsically disordered regions (IDRs). Despite this structural complexity, the Ubp3/Bre5 complex could be isolated following either co-expression in vivo or after in vitro assembly. Unexpectedly, complex formation with Bre5 was not required for the catalytic activity of full length Ubp3. Furthermore, even the isolated catalytic domain of Ubp3 was fully active against two distinct substrates in the absence of Bre5, demonstrating that its deubiquitinating activity is intrinsically independent of Bre5. These findings indicate that the catalytic domain alone is sufficient for substrate cleavage, whereas the extensive IDRs of Ubp3 and its cofactor Bre5 might contribute to substrate recognition or specificity. Overall, this study challenges the prevailing model of Bre5-dependent activation of Ubp3 and provides new insights into the molecular organization of the Ubp3/Bre5 system. More broadly, it highlights the importance of intrinsically disordered regions in regulating deubiquitinase function and cellular signaling networks.

Mutations in mitochondrial DNA (mtDNA) compromise ETC activity and impair oxidative phosphorylation. Since eukaryotic cells contain multiple copies of mtDNA, the resulting phenotype depends on the proportion of mutant mitochondrial genomes (the heteroplasmy level). Using isogenic cell lines carrying similar mtDNA deletions, a linear decline in cellular respiration was observed as mitochondrial DNA heteroplasmy increased. Despite this, cellular redox imbalance did not change until heteroplasmy exceeded 50%. As heteroplasmy increased past 70%, cells also exhibited an integrated stress response (ISR) and impaired translation was observed. These defects were reversed by either addition of asparagine or overexpression of pyruvate carboxylase (PC). The dependence on exogenous asparagine in other respiration-deficient cells was found to correlate inversely with the PC expression level. For example, patient-derived thyroid tumor cells, harboring high heteroplasmy for a Complex I mtDNA mutation and low levels of PC, exhibited asparagine auxotrophy, and L-asparaginase treatment suppressed tumor growth. Together, these findings demonstrate a role for mitochondrial pyruvate carboxylase in cellular asparagine synthesis under conditions of compromised respiratory activity.

Uncoupling protein 1 (UCP1) mediates thermogenesis in brown adipose tissue. Whether human-UCP1 shares the bioenergetic properties established for rodent UCP1 (innate uncoupling, GDP sensitivity, fatty acid (re)activation) is not known. Therefore, we expressed human and mouse UCP1 in mouse liver, using adeno-associated viral vectors, and characterized their properties in isolated liver mitochondria. Both UCP1s induced marked innate uncoupling, characterized by increased substrate-supported respiration and decreased membrane potential, in the absence of exogenous fatty acids. Mouse-UCP1 in liver retained the classical regulatory properties of native brown-fat UCP1, including potent inhibition by GDP and reactivation by oleate. In contrast, human-UCP1 was only weakly inhibited by GDP but was strongly responsive to fatty acids. However, ATP potently inhibited human-UCP1, with an apparent IC of {approx}0.4 mM compared with {approx}1.4 mM for GDP, and ATP markedly decreased the sensitivity of human-UCP1 to oleate (re)activation. Despite substantial UCP1-mediated uncoupling, oxidative phosphorylation capacity and mitochondrial OXPHOS protein levels were preserved. Molecular dynamics simulations suggested a structural basis for the species difference. GDP formed persistent interactions with F88 in mouse-UCP1, an interaction absent at the corresponding S88 residue in human-UCP1. In-silico substitution of F88 by serine reduced GDP interaction at this site. Thus, human and mouse UCP1 share innate thermogenic activity but differ fundamentally in nucleotide regulation. The F88/S88 difference may contribute to the preferential GDP sensitivity of mouse-UCP1, whereas ATP provides effective nucleotide control of human-UCP1.

AbstractPhospholipid asymmetry is a hallmark of mammalian cell membranes and reflects the selective distribution of distinct phospholipid species between the two leaflets of the lipid bilayer. Although this asymmetry is tightly maintained, the membrane proteins whose functions depend on it remain largely unknown. To perturb phospholipid asymmetry experimentally, we expressed a constitutively active phospholipid scramblase and thereby identified transient receptor potential melastatin 8 (TRPM8) as an ion channel regulated by this membrane property. Activation of TRPM8 by both l-menthol and innocuous cold was markedly suppressed following disruption of phospholipid asymmetry. Likewise, selective depletion of phosphatidylserine (PS), a phospholipid enriched in the cytoplasmic leaflet, using a cytosolically targeted PS decarboxylase attenuated TRPM8 activation, indicating that cytoplasmic PS is required for proper TRPM8 function. Mechanistically, our findings suggest that cytoplasmic PS supports efficient TRPM8 activation by maintaining the biochemical state of the channel. Together, these findings identify TRPM8 as a phospholipid asymmetry-dependent ion channel and establish an experimental strategy for systematically identifying membrane proteins regulated by phospholipid asymmetry. This work provides a foundation for future studies investigating the biological significance of this fundamental membrane property.

Nitrite can be reduced to nitric oxide (NO) by several heme- and molybdenum-containing proteins, including mitochondrial cytochrome c oxidase (Cco). This activity, designated Cco/NO, has been implicated in hypoxic signaling, but its regulation and quantitative significance relative to other NO-producing systems remain uncertain. We examined its modulation by adenine nucleotides using detergent-solubilized yeast and mouse brain mitochondria supplied with 1 mM nitrite and an ascorbate/TMPD/cytochrome c electron-donor system. ADP and ATP differentially modulated Cco/NO activity, and ADP extended measurable NO formation across the entire oxygen range tested, up to the assay ceiling of 175 {micro}M O2. Nucleotide regulation was also isoform-dependent: ATP slightly inhibited Va-containing Cco but strongly stimulated Vb-containing Cco under anoxic conditions. Rates normalized to cytochrome aa demonstrate multi-turnover nitrite-reductase capacity under these substrate-driven assay conditions. Both the cellular ADP/ATP ratio and subsequently assayed Cco/NO activity increased transiently following a hypoxic shift. These findings establish metabolic and isoform-dependent gating of the catalytic capacity of Cco/NO; they do not establish its fractional contribution to total cellular NO or its operation at physiological nitrite concentrations in intact, coupled mitochondria. This research was supported by CONICET Grant PIP 706 (research team member P.R.C.) and National Institutes of Health Grant GM30228 to R.O.P.

Lipid droplets (LDs) are unique organelles, surrounded by a phospholipid monolayer. They are present in most eukaryotic cells including the unicellular model organism S. cerevisiae. LDs store neutral lipids which serve as precursors for amphipathic membrane lipids and as an energy reserve. Loss of LDs in S. cerevisiae results in multiple cellular defects impairing lipid homeostasis and the biogenesis and function of other organelles. Here, we find that the expression levels of many proteins in isolated mitochondrial fractions are altered in cells that cannot synthesize neutral lipids and therefore lack LDs. In addition, among several downregulated proteins, we identified the previously uncharacterized Ylr001c (which we name Vlf1 for Vacuolar Lipophagy Factor 1). We show that Vlf1 is glycosylated and, in contrast to some previous reports, is actually localized to the vacuole. Furthermore, we demonstrate that changes in Vlf1 expression alter growth sensitivity to rapamycin, and detected a physical interaction of Vlf1 with Atg15, a lipase involved in autophagy. Additionally, we observe higher levels of autophagy/lipophagy in the absence of Vlf1 and a reduction upon overexpression of the protein. Taken together, the effects on lipohagy by Vlf1 makes it, according to our knowledge, the first vacuolar lipophagy regulator identified in S. cerevisiae.

THAP9 is a transposable element-derived gene which encodes a protein that is homologous to the active Drosophila P-element transposase (DmTNP). Both THAP9 and DmTNP possess a C-terminal domain (CTD) which is functionally uncharacterized. Sequence and structural analysis suggest that the THAP9-CTD has a novel fold which is only found in THAP9 homologs. To explore the evolutionary history and characteristics of this novel domain, exhaustive phylogenetic analysis (using MSA, structure prediction, MSTA-based clustering) was performed. THAP9-CTD homologs were more widely distributed throughout the animal kingdom in comparison to DmTNP-CTD homologs which were restricted to arthropods. Moreover, the THAP9-CTD homologs were more conserved, especially among mammals and birds and their average length increased in a class-specific manner. Comparison with the DmTNP-CTD homologs demonstrates that although their respective CTDs may have evolved independently, they both surprisingly share similar secondary structure elements consisting of three conserved helical regions made of hydrophobic residues that are predicted to make up a conserved core. The role of the respective CTDs were further investigated by creating truncation mutants lacking the CTD. Interestingly both THAP9 and DmTNP truncation mutants are still capable of DNA excision and integration suggesting that their respective CTDs are not essential for DNA transposition. Moreover, CTD truncation favours DNA integration in THAP9: this suggests that CTD acquisition during evolution may have led to THAP9 domestication as observed in other transposable element-derived genes like Rag1 and piggybac, which have similar terminal regulatory domains.

Lysophosphatidic acid (LPA) is widely recognized as an extracellular lipid mediator; however, the functional significance of intracellularly produced LPA remains poorly understood. Here, we investigated the regulatory mechanism and functional role of a LPA-producing lysophospholipase D GDE4, also known as GDPD1, in prostate cancer cells. GDE4 expression is induced under ER stress conditions in a PERK-dependent manner and requires the transcription factor ATF3. Disruption of GDE4 expression resulted in altered intracellular levels of LPA and LPA precursor lysophosphatidylethanolamine, accompanied by reduced cell proliferation. RNA sequencing and subsequent validation identified a set of genes downregulated in GDE4-depleted cells. Pharmacological inhibition experiments indicated that peroxisome proliferator-activated receptor and {gamma} (PPAR and PPAR{gamma}) signaling pathways contribute to the regulation of these GDE4-dependent genes. Collectively, our findings suggest that GDE4-dependent lipid remodeling is associated with PPAR/{gamma}-mediated transcriptional regulation under ER stress conditions. These results provide a potential framework for understanding the link between intracellular lipid metabolism and stress-responsive gene regulation.

Endosomal trafficking is a major pathway that delivers cell-surface proteins, including glutamate receptors, to support neurotransmission and normal brain functions. Activity-dependent insertion of glutamate receptors is essential for synaptic plasticity, learning and memory. Copine-6 is a neuronal-specific calcium (Ca2+) binding protein that mediates activity-induced exocytosis of -amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)-type glutamate receptors. The activation of N-methyl-D-aspartate (NMDA) receptors triggers Ca2+-dependent translocation of Copine-6 to intracellular endosomal compartments. However, the mechanisms underlying the activity-dependent accumulation of Copine-6 in endosomes remain unknown. Here, we show that Copine-6 exhibits Ca2+-dependent binding to phosphatidylinositol-3-phosphate (PI(3)P) through the C2B domain and displays enhanced interaction with active Rab11a in a Ca2+-independent manner via the vWA domain. Mutations in the C2B that inhibit binding to PI(3)P not only block the activity-induced translocation of Copine-6 to early endosomes, but it also causes an aberrant accumulation of Copine-6 in recycling endosomes. Consequently, loss of Copine-6 expression impairs the efficient coupling of early and recycling endosomes and blocks activity-dependent delivery of both AMPA and NMDA receptors onto the neuronal plasma membrane. These defects can be restored by re-expressing wild-type Copine-6, but not the C2B phospholipid-binding mutant. Together, our findings establish Copine-6 as a molecular bridge that enhances coupling between the early and recycling endosomal membranes, thereby facilitating the activity-dependent forward trafficking of glutamate receptors to the neuronal plasma membrane to maintain synaptic potentiation.

Cyclic adenosine monophosphate (cAMP) is a second messenger that regulates various cellular processes, including the activity of hyperpolarization-activated channels (HCN), which are implicated in cardiac physiology and neurodegenerative diseases such as Parkinsons disease (PD). In this study, we used a photoactivated adenylyl cyclase (PAC) S27A mutant to optogenetically control intracellular cAMP levels. We demonstrated that light-induced elevation of cAMP activated HCN4 channels, leading to increased beating rates in cardiomyocytes. Unilateral expression of PAC(S27A) in the substantia nigra pars compacta of mice induced rotation behavior upon light stimulation, which could be attenuated by HCN inhibitors. Furthermore, PAC(S27A) activation partially recovered motor deficits in a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced PD mouse model, accompanied by increased HCN2 channel expression in ipsilateral basal ganglia. Our findings highlight the potential of using optogenetics to modulate cAMP and HCN channel activity for the treatment of cardiac and neurological disorders.

Blue light using Flavin (BLUF) proteins are microbial photoreceptors that are involved in various physiological responses. Their occurrence and biochemical properties in fungi remain poorly understood. Here, we investigated a putative BLUF photoreceptor from the corn-smut fungus Mycosarcoma maydis (MmBLUF). Domain analysis, multiple sequence alignment of BLUF core regions, and structural modelling indicated conserved canonical BLUF fold and flavin-pocket residues. However, when heterologously expressed, UV-visible and fluorescence spectroscopy revealed different spectral behaviour than canonical BLUF protein. Further, we tested the role of extended N-terminus in modulation of chromophore binding by expressing N-terminus truncated protein variants. Our results suggest that the unusual spectral behaviour is not linked to the truncation construct (extended N-terminus), which also showed similar spectral features, indicating that the extended N-terminus is unlikely to account for an unusual photodynamics characteristics. Our findings support MmBLUF as a structurally conserved putative fungal BLUF-like photoreceptor with different photochemical properties. Further studies are required to establish its chromophore identity, photocycle and function of this unusual BLUF-like domain from fungal system.

Melittin exhibits antitumor activity in cervical cancer models, yet the long non-coding RNA (lncRNA) response and associated regulatory networks remain poorly understood. Here, strand-specific RNA-seq data from melittin-treated and untreated U14 murine cervical cancer cells were analyzed to characterize melittin-responsive lncRNAs and explore their potential functional associations. A total of 28,162 lncRNAs were identified, including 27,307 known and 855 novel transcripts. Differential expression analysis revealed 404 differentially expressed lncRNAs (DElncRNAs), comprising 191 upregulated and 213 downregulated lncRNAs, w most of which were predicted to localize to the cytoplasm or nucleus. Cis-target analysis identified 52 neighboring mRNAs as putative targets of 46 DElncRNAs. Functional enrichment highlighted mitochondrial electron transfer and redox-related processes, including the mitochondrial electron transfer flavoprotein complex, electron-transferring-flavoprotein dehydrogenase activity, ubiquinone binding, and quinone binding. In parallel, melittin induced mitochondrial membrane depolarization and increased intracellular reactive oxygen species accumulation in U14 cells. Co-expression analysis further identified 138 lncRNAs co-expressed with 161 mRNAs, which were enriched in chromatin remodeling, DNA replication, and DNA repair. EdU incorporation decreased with increasing melittin concentrations, indicating suppression of DNA synthesis and proliferative activity. RT-qPCR analysis confirmed the expression trends of selected DElncRNAs. Collectively, these findings demonstrate extensive remodeling of the lncRNA landscape in melittin-treated U14 cells and suggest that melittin-responsive lncRNA-mRNA networks are associated with mitochondrial redox disruption and impaired DNA synthesis. This study provides a transcriptomic framework for identifying candidate lncRNA-mRNA regulatory axes underlying the antitumor response to melittin.

GPR27/SREB1 is a highly conserved orphan class A G-protein coupled receptor implicated in insulin production, metabolic regulation, tumour biology, neurodegeneration and L-lactate homeostasis, but its immediate second-messenger signalling remains poorly defined. We used single-cell Forster resonance energy transfer nanosensors to monitor cytosolic Ca2+ and cAMP in wild-type 3T3 MEF cells, CRISPR-Cas9 GPR27-knockout cells (GPR27KO) and GPR27-knockout cells transiently re-expressing FLAG-tagged GPR27 (GPR27-rescued). The GPR27 surrogate agonist 8535n (1 {micro}M) increased intracellular Ca2+ in wild-type and rescued cells but not in GPR27-knockout cells and produced no significant cAMP response in wild-type cells. Basal Ca2+ and cAMP levels were unaffected by GPR27 deletion. Extracellular L-lactate (2 mM) induced a GPR27-dependent increase in Ca2+ and cAMP in wild-type and rescued cells, but not in knockout cells, raising the possibility that L-lactate acts as an endogenous ligand or modulator of GPR27. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=111 SRC="FIGDIR/small/743761v1_ufig1.gif" ALT="Figure 1"> View larger version (10K): org.highwire.dtl.DTLVardef@2af45dorg.highwire.dtl.DTLVardef@113e2corg.highwire.dtl.DTLVardef@8dea4org.highwire.dtl.DTLVardef@50ec49_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIGPR27 surrogate agonist 8535n increases intracellular Ca2+ but not cAMP in 3T3 cells. C_LIO_LIExtracellular L-lactate induces GPR27-dependent intracellular Ca2+ and cAMP increases in 3T3 cells. C_LIO_LIThese findings identify GPR27 as a putative candidate lactate sensor. C_LI

Cell confluency is a major determinant of cell-cell communication, protein interactions, access to nutrients, and signalling dynamics, thereby significantly impacting biological outcomes. Lung cancer cells like A549 are widely used as screening models for scientific studies wherein their growth in vitro progress from non-confluent to confluent growth. In this study, we investigated the transcriptomic adaptations associated with the transition of A549 cells from baseline non-confluent to confluent growth. Comparative transcriptomic analysis between confluent and cells at baseline identified 815 upregulated and 671 downregulated transcripts. Pathway enrichment analysis of deregulated transcripts in confluent cells revealed enhanced cholesterol and sterol biosynthetic pathways, along with suppression of chromosomal segregation and mitotic pathways. At confluency, an increased expression of glucose transporters (SLC2, SLC60, and SL37 families) and glycolytic pathways, and a decrease in amino acid transporters (SLC1, SLC7, SLC38, and SLC36) and amino acid metabolic pathways is observed. A reduced one-carbon metabolic signature (SHMT2, DHFR, and MTHFD2) and enhanced fatty acid precursor synthesis (HMGCLL1, ALDH6A1, and AASS) were also observed at confluency. 1H NMR profiling of culture media revealed higher glucose and glutamine utilisation with lactate accumulation during culture maturation. Collectively, the data suggest transcriptome-level rewiring in A549 cells with preferential biosynthesis of lipids and sterols at confluency and underscore the importance of considering culture maturity in cancer biology, metabolism, and therapeutic studies.

Collective cell migration is a fundamental process driving tissue repair, angiogenesis, and vascular homeostasis. This coordinated movement requires both intercellular communication via gap junctions and precise intracellular Ca{superscript 2} signaling, largely regulated by the sarco(endo)plasmic reticulum Ca2+ ATPase (SERCA) pump within the endoplasmic reticulum (ER). Historically, carbenoxolone (CBX)--a synthetic derivative of glycyrrhetinic acid--has been widely utilized as a pharmacological tool to inhibit gap junctions and dissect their role in collective cell motility. However, its molecular specificity remains highly controversial. In the present study, using different cellular models, we found that CBX drastically reduces collective cell migration by a previously undescribed function for CBX: a fast, potent, and reversible inhibition of the SERCA pump, which provokes a passive leak of the luminal ER Ca{superscript 2} store. Our findings suggest that the effect of CBX over many cellular responses including cell migration and communication, previously only attributed to gap junction blockade, are indeed the consequence of the disruption of intracellular Ca{superscript 2} homeostasis. One Sentence Summarycarbenoxolone blocks cell migration by inhibiting SERCA

A pair of paralogs, Chchd3 and Chchd6, two components of mitochondrial contact site and cristae organizing system (MICOS), have been identified to be candidate pathogenetic genes in congenital heart disease (CHD). Previous research found that knockdown (KD) of the single Chchd3/6 (Chchd3) gene and other MICOS components in Drosophila impaired heart function, likely due to a deficit in mitochondrial organization, ATP production, actomyosin levels, and thus severely diminished contractility. However, the underlying mechanisms of how MICOS deficiency leads to these defects are not clear. Here, we performed genetic manipulations in the Drosophila heart to probe for possible interactions between MICOS-compromised mitochondria and other organelles and processes. We found that moderate reduction in Pink1/parkin-mediated mitophagy synergistically aggravated cardiac Chchd3 KD phenotypes, indicating a major interaction. Further, Chchd3 KD increased the level of reactive oxygen species (ROS) and endoplasmic reticulum (ER) stress. Interestingly, KD of catalase (CAT) also elevated cardiac ROS levels, but surprisingly did not compromise contractility either by itself or in combination with Chchd3 KD to aggravate the cardiac phenotype. However, CAT overexpression (OE) in Chchd3 KD hearts restored contractility, but only partially, even though elevated ROS due to Chchd3 KD was fully normalized. Similarly, counteracting ER stress by overexpressing Xbp1 (or spliced mouse Xbp1) also partially rescued the heart function defects induced by Chchd3 KD. Overall, these data indicate a critical role of mitophagy and ER/oxidative stress in cardiac homeostasis involving Chchd3, which suggests that deficiency of MICOS function contributes to heart dysfunction via multiple stress responsive pathways.

Cholesterol efflux is an important determinant of cellular lipid homeostasis. However, how microglial excessive cholesterol accumulation affects neuronal synaptic integrity remains poorly understood, particularly in the context of Alzheimer's disease. Here, we utilized a conditional knockout mouse model targeting the cholesterol transporters ABCA1 and ABCG1 in microglia. The microglia-specific ABCA1/ABCG1 deficiency triggered marked cholesterol accumulation, microglial hypertrophy, downregulation of the homeostatic marker P2ry12, and upregulation of the reactivity-associated marker CD11b, indicating shift toward a reactive phenotype. This phenotype was accompanied by increased reactive oxygen species, consistent with enhanced oxidative stress in ABCA1/ABCG1-deficient microglia compared with control. Using organotypic hippocampal slice cultures, we investigated the downstream neuronal outcomes of microglial ABCA1/ABCG1 deficiency. Under basal conditions, microglial ABCA1/ABCG1 knockdown did not significantly alter dendritic spine density in CA1 pyramidal neurons. However, upon exposure to amyloid-beta (A{beta}) stress, microglial ABCA1/ABCG1 deficiency markedly exacerbated dendritic spine loss in CA1 pyramidal neurons. Taken together, our findings highlight an important role for ABCA1/ABCG1-dependent cholesterol efflux in maintaining microglial homeostasis and limiting neuronal synaptic vulnerability to A{beta}-associated stress. These results support further investigation of microglial cholesterol transport as a potential target for preserving synaptic resilience in Alzheimer's disease.

The perilipins generally represent the most abundant lipid droplet (LD) surface proteins in mammalian cells and can target LD subpopulations within the same cell. They are characterized by a conserved organization of disordered and folded regions, as well as a number of divergent features, which contribute to differences in perilipin function and LD targeting. Here, we focus on the C-terminal 4-helix bundle (4HB) domain that is present in all perilipins except for PLIN1. Using biochemical and in silico approaches, we show that the 4HB of PLIN3 is a stably folded domain and interacts with lipid surfaces in vitro and with LDs in model cells. The {beta}-subdomain at the bottom of the helical bundle is required for the binding to LDs, but not for the 4HB stability, suggesting that this region may promote direct interaction with the LD surface. In agreement, the 4HB of PLIN4, which does not contain an {beta}- subdomain, does not bind to LDs. Overall, our work shows that small differences in perilipin structural features impact their differential targeting to LDs.

Biomolecular condensates are membraneless compartments critical for the functional organization of cellular macromolecules in essential processes such as cell division, gene transcription or stress responses. We previously reported that vimentin filaments remodel into phase separated biomolecular condensates upon oxidative stress. This process requires vimentin single cysteine, C328, suggesting the involvement of oxidative modifications of this residue. Here, we aimed to generate vimentin condensates by inserting mutations mimicking posttranslational modifications associated with oxidative stress. In vimentin deficient cells, a cysteine oxidation mimetic mutant, vimentin C328D, formed only elongated particles or short filaments that evolved towards droplets upon serum deprivation or treatment with the oxidant diamide. Among vimentin posttranslational modifications rapidly responding to these stimuli, glycosylation confers filament stability whereas phosphorylation promotes disassembly. We observed that the O-deglycosylation inhibitor thiamet G, and the kinase inhibitors staurosporine and H-89, attenuated diamide-elicited vimentin C328D droplet formation, suggesting a potential glycosylation/phosphorylation interplay in this effect. Indeed, introducing phosphomimetic residues at certain single vimentin glycosylation and/or phosphorylation sites induced the formation of droplets, only if combined with the C328D mutation. In particular, the vimentin S49D,C328D mutant formed condensates that were reversibly dispersed by dilution through hypotonic shock. Therefore, mimicking C328 oxidation and S49 phosphorylation was sufficient to elicit vimentin phase separation. In vitro, purified vimentin S49D,C328D polymerized into a mixture of aberrant filaments and aggregates, which, in the presence of crowders, evolved towards paracrystals or clusters of beaded assemblies depending on pH. These findings highlight the role of C328 perturbations in the formation of biomolecular condensates and suggest a modulatory role of glycosylation/phosphorylation, thus shedding light on the processes regulating vimentin phase separation.

Orthodontic tooth movement relies on a tightly regulated pro-inflammatory and pro resorptive mechanoresponse of local periodontal ligament fibroblasts (PdLFs). Dysregulation is linked to complications such as root resorption and tooth loss. Hyperlipidemic conditions promote excessive PdL mechanoresponses, with growth differentiation factor 15 (GDF15) acting as potential regulator. This study examined the contribution of the inflammasome/pyroptosis pathway as underlying mechanism for dysregulated mechanoresponses. Human PdLFs were treated with palmitic acid (PA) or oleic acid (OA) for six days before 24 hours of compressive loading. PA increased CASP1, CASP4, and CASP3 activity, secretion of IL-1{beta}, IL-18, and HMGB1, and LDH release. Pharmacological blockade and siRNA-mediated knockdown of inflammasome- and pyroptosis-related targets revealed that NLRP3, CASP1, CASP4, and GSDMD partially contributed to monocyte and osteoclast overactivation. Silencing PA-increased GDF15, partially normalized the phenotype, at least in part by inflammasome/pyroptosis regulation. GDF15 acted through extracellular, and a nuclear signaling route, each accounting partially to this phenotype. Together, GDF15 partially regulates the PA-induced, pyroptosis-associated overactivated mechanoresponse alongside pyroptosis-independent mechanisms suggesting it as an interesting target for potential clinical interventions.