Life Science Alliance

Special AT-rich binding protein 1 (SATB1) is a nuclear matrix-associated transcription factor that orchestrates higher-order chromatin architecture and gene expression in T and B lymphocytes. While SATB1s role in T cells is well-established, its regulation and function in B cells remain underexplored. Here, we showed that Satb1 expression in mature B cells is controlled by alternative promoter usage and splicing, with promoters P1 and P3 dynamically switching upon LPS-induced activation, while P2 remains transcriptionally silent. We identified four Satb1 mRNA isoforms, including two novel transcripts ({Delta}9 and{Delta} 9{Delta}11), all of which maintain open reading frames. Satb1 transcription decreases upon activation while SATB1 protein content evolves differently, suggesting post-transcriptional regulation. SATB1 forms a high-molecular-weight complex in B cells, and co-immunoprecipitation mass spectrometry reveals RNA-binding proteins as its predominant interactors. These findings specify SATB1 expression kinetics during B-cell activation and reveal its role as a regulator of RNA splicing in B cells.

RASAL3 acts as a negative regulator of small cellular GTPases in hematopoietic cells. In immune cells, it primarily modulates the RAS/MAPK signaling pathway and affects cellular events including proliferation, differentiation, survival, and migration. Due to its inhibitory role in T cells, RASAL3 may represent a potential modulatory target for improving therapeutic strategies such as cell-based immunotherapy. However, most existing knowledge about RASAL3 function is derived from murine models, and its role in human T-cell signaling remains insufficiently characterized. To address this gap, we systematically investigated the function of RASAL3 in human primary T cells and T-cell line. For this purpose, we employed RASAL3 overexpression, CRISPR/Cas9-mediated deletion, and siRNA-mediated knockdown to thoroughly analyze the effects of RASAL3 on T-cell signaling, proliferation, and migration. Our data demonstrate that RASAL3 modulates primarily CDC42 and RAC1/RAC2 GTPases activity, SAPK/JNK phosphorylation, c-Fos and c-Jun expression, and IL-2 gene promoter activation. In addition, RASAL3 regulates actin polymerization and T-cell migration. Notably, loss of RASAL3 increases Jurkat T cells motility in vivo and potentiates their homing to the spleen. Collectively, these findings identify RASAL3 as an important regulator of human T-cell activation and motility and highlight its application potential for improving CAR-T cell therapy.

Directional cell migration requires cells to sense extracellular cues and coordinate adhesion, cytoskeletal remodelling and polarity, yet the molecular regulators that integrate these events remain incompletely defined. PTTG1-binding factor (PBF/PTTG1IP) is a transmembrane glycoprotein extensively characterised in pathological overexpression and cancer models, where elevated expression promotes tumourigenic cellular phenotypes. However, its endogenous physiological function remains poorly understood. Here, separate enrichment analyses of transcriptomic and phosphoproteomic profiles from PBF-overexpressing cells collectively highlighted cell adhesion, extracellular matrix organisation, cytoskeletal regulation and cell motility as major PBF-associated programmes. Using a novel Pbf knockout mouse model, we show that primary Pbf-KO mouse embryonic fibroblasts exhibit impaired migration and invasion. PBF loss reduced fibronectin adhesion, disrupted focal adhesion number, size and distribution, attenuated FAK Tyr397 phosphorylation and delayed early adhesion formation. Live-cell imaging and Golgi orientation assays further revealed altered actin organisation and impaired front-rear polarity in Pbf-KO cells. Key phenotypes were conserved in CRISPR-Cas9 PBF-KO human thyroid cancer cells, while targeted PBF re-expression restored migration in Pbf-KO MEFs. We thus identify PBF as an endogenous regulator of cell adhesion, polarity and directional motility, providing a framework for understanding how pathological PBF overexpression may promote invasive behaviour in cancer.

NF-{kappa}B essential modulator (NEMO) is the critical scaffold protein of the canonical I kappa B kinase (IKK) complex. Whilst studying the contribution of IKK and IKK{beta} to NF-{kappa}B signalling we noted that NEMO protein levels were reduced in IKK knockout (KO) HCT116 cells. Loss of NEMO was more pronounced in IKK KO cells than IKK{beta} KO, whilst IKK+IKK{beta} double knockout (DKO) cells exhibited a 90% reduction in NEMO abundance. In contrast, the abundance of IKKs was unaffected by NEMO KO, although this abolished canonical NF-{kappa}B activation. Loss of IKKs had no effect on NEMO mRNA levels but IKK DKO cells exhibited accelerated NEMO protein turnover that was reversed by the proteasome inhibitor, MG132, but not the NEDDylation inhibitor, MLN4924. TAK-243, a selective inhibitor of UBA1, the E1 ubiquitin-activating enzyme, increased basal NEMO levels in DKO cells but did not prevent NEMO turnover suggesting both ubiquitylation-dependent and independent pathways for degradation. Bioinformatic analysis identified substantial regions of intrinsic disorder within the N- and C-termini of NEMO. NEMO was rapidly degraded by isolated 20S proteasomes in vitro and this was inhibited by interaction with IKK or IKK{beta}. Finally, re-expression of IKK increased NEMO expression in IKK DKO cells and this required the C-terminal NEMO binding domain (NBD) but not a functional kinase domain. Thus the IKKs act as critical kinase-independent scaffolds that stabilise NEMO; loss of these interactions results in NEMO degradation via the proteasome. These results may be relevant to strategies for inhibiting NF-{kappa}B signalling by disrupting NEMO:IKK interactions.

Lamins are nuclear intermediate filament proteins that maintain nuclear architecture through interactions with the chromatin. The A- and B-type lamins tether the genome at the peripheral lamina underlying the inner nuclear membrane in the form of heterochromatic lamina-associated domains (LADs). However, LADs associated with A-type lamins are confined not only to lamina but also to the nuclear core, thereby pointing to their distinct and multifarious roles in chromatin organisation and regulation. Previous studies involving lamin B1 depletion depicted the detachment of LADs from the nuclear periphery, accompanied by alteration of chromatin distribution while preserving the topologically associating domains (TADs) in structurally intact form. In this piece of work, we have shown, for the first time in HGSOC, the effects of lamin A/C knockdown on chromatin organisation, which was characterised by significant detethering of gene-poor chromatin from the periphery along with active (A) to inactive (B) compartment switching. This was associated with a rewiring of oncogenic super-enhancer elements corroborated by the differential gene expression profile. Overall analysis tipped the scale in favour of reduced cellular proliferation upon lamin A/C knockdown. This finding was strengthened by the observed proliferative potential of spheroids ex vivo and tumours in a mouse xenograft model.

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.

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.

Microtubules (MTs) undergo continuous cycles of growth and disassembly. Because the transitions between these states are stochastic, MT age varies widely within a population. As MTs age, they are thought to accumulate post-translational modifications (PTMs) that, directly or indirectly, enhance their stability and thereby extend their lifetime. The rare MTs that withstand prolonged exposure to destabilizing drugs such as nocodazole (NZ) are indeed enriched in PTMs; yet the relationships between MT age, PTMs and stability remain unclear. Using microinjection of labelled tubulin, we measured microtubule network turnover in immortalized mouse embryonic fibroblasts. Half of the network was renewed within 3 minutes and 80% within 10 minutes, while approximately 5% of microtubules persisted for more than 20 minutes. These dynamics were comparable in quiescent and senescent cells, although the fraction of slowly renewing or non-renewing microtubules rose to 20% in senescent cells. Unexpectedly, neither the amount of PTMs (acetylation and detyrosination) nor resistance to NZ increased with MT age, and resistance to NZ was independent of these PTMs. Degrees of acetylation and detyrosination should therefore not be taken as readouts of MT age or stability. Because these PTMs do not accumulate on MTs over time, the chemical modification of polymerized tubulin is likely more reversible and dynamic than previously assumed.

The QxxR motif is evolutionarily conserved within DEAD-box RNA helicases, including Drosophila Me31B and human DDX6, which post-transcriptionally regulate gene expression during animal development. A pathogenic H372R substitution (QxHR to QxRR) in the QxxR motif of human DDX6 has been associated with various developmental defects, but how this motif contributes to DDX6-family protein function remains unclear. Here, we used Drosophila Me31B as an in vivo model to investigate the QxxR motifs developmental role. We generated a Drosophila strain carrying the corresponding H333R missense mutation in Me31B and characterized its effects on female fertility, embryonic viability, germline development, and Me31B-associated molecular pathways. The me31BH333R mutation reduced female fertility in a gene dose-dependent manner, with homozygous mutant females being sterile. Embryos from the mutant females also exhibited primordial germ cell defects. Despite these developmental phenotypes, the me31BH333R mutation did not significantly alter Me31B protein abundance, global ovarian transcriptome or proteome profiles, or representative germ plasm mRNA and protein localization. In contrast, bait-normalized IP-MS analysis revealed altered enrichment of selected Me31B-associated proteins, including increased association of known Me31B interactors Trailer hitch (Tral) and Ypsilon Schachtel (Yps). These findings establish Me31BH333R as an in vivo model for investigating the conserved QxxR motif and suggest that disruption of this motif compromises development not through broad changes in gene expression, but potentially through altered composition or regulation of Me31B-containing ribonucleoprotein complexes.

SUMOylation is an essential eukaryotic ubiquitin-like post-translational modification that plays a central role in the regulation of various nuclear processes and stress responses. It canonically occurs at lysine residues within {Psi}KXE consensus motifs that lie in intrinsically disordered regions or loops and interact specifically with the SUMO-conjugating E2 enzyme UBC9. However, many detected SUMOylation sites are found within structured domains, and it remains unclear how these are recognised by UBC9. Here, we investigated the SUMOylation of Lys43 in the BTB domain of human ZBTB38 (ZBTB38BTB), a lysine located within a rigid {beta}-sheet. By combining X-ray crystallography, structural prediction, and in-vitro UBC9 interaction and SUMOylation assays, we show that ZBTB38BTB possesses a dedicated surface that recapitulates the spatial arrangement of residues found in canonical linear consensus motifs. This surface binds UBC9 with mid-micromolar affinity and is predicted to position Lys43 in its active site for efficient SUMOylation. Structural modelling and sequence analyses suggest that this property is shared by BTB domains of five members (10%) of the ZBTB-protein family across vertebrates, revealing a previously unrecognised property of a subset of ZBTBBTB domains. Kinetic analyses reveal that, under the reaction conditions used, the catalytic efficiency of ZBTB38BTB and ZBTB33BTB SUMOylation are closely comparable to that of the C-terminal domain of RANGAP1, the best-characterised and most efficiently SUMOylated substrate known. This defines a new class of inherently efficient, E3 ligase-independent SUMOylation substrates beyond RANGAP1 and suggests that structural pre-organisation of the acceptor lysine and its environment may promote productive UBC9 engagement. Lastly, we demonstrate the presence of higher-molecular-weight, modified forms of ZBTB38 in human cells, consistent with SUMOylation. Together, these results provide a biochemical basis for interpreting existing and designing future studies on the functional impact of ZBTB SUMOylation. More broadly, our findings offer insights into the determinants of efficient SUMOylation, and may facilitate the identification of further inherently efficient targets, and, potentially, the design of SUMOylation modulators.

Accurate and tightly coordinated cell cycle progression and cell proliferation are critical for development, growth and homeostasis of an organism. Recently, Zinc finger protein 768 (ZNF768) was identified as a transcription factor driving cellular proliferation, in both a p53-dependent and independent manner. ZNF768 interacts with and represses p53 functions to limit cell cycle delay. Independently, ZNF768 promotes the transcription of key regulators of the cell cycle machinery, although the mechanisms through which this occurs remain unknown. Here, we report that ZNF768 protein levels are tightly regulated during the cell cycle, and its depletion leads to cell cycle exit and induction of quiescence. We found that ZNF768 modulates the cell cycle, at least in part, by controlling expression of the major pro-proliferative transcription factor E2F1 independently of p53 activation. Consequently, depletion of ZNF768, which also represses expression of the key mitotic transcription factor and E2F1 target FOXM1, leads to numerous mitotic errors. Supporting these findings, cancer genomics analyses reveal that ZNF768 expression levels are positively associated with E2F1 and FOXM1 expression levels in human tumors, suggesting that cancer cells might use ZNF768 to override cell cycle arrest, sustain proliferation, and promote cancer progression. Altogether, our results reveal that ZNF768 modulates cell cycle entry and proliferation, at least in part by regulating E2F1 expression.

The differentiation of naive CD4+ T cells into effector T cell subsets such as TH1, TH2, TH17 and TREG cells is governed by tightly coordinated activation programmes dependent on T cell receptor (TCR) engagement, co-stimulation, and cytokine signalling. This differentiation process involves regulation of stress-responsive signalling pathways such as the unfolded protein response (UPR) and the oxidative stress response (OSR) to adapt to the physiologic demands specific to distinct T cell subset functions. This suggests that pharmacologically targeting stress-responsive pathways offers a unique opportunity to selectively remodel the differentiation of these different T cell subsets. We previously identified compound AA147 as a metabolically activated proteostasis regulator that can induce both the ATF6 signalling arm of the UPR and, in certain cell types, the NRF2-regulated arm of the OSR. (Paxman et al., 2018; Plate et al., 2016) Here, we show that treatment with AA147 selectively reduces differentiation of pro-inflammatory TH17 cells by promoting degradation of the lineage-specifying transcription factor RORgammat, without impacting the transcription factors of other CD4+ T cell subsets. AA147-dependent reduction in TH17 differentiation is independent of ATF6 activation and involves activation of NRF2, which reduces intracellular reactive oxygen species (ROS) to hinder TH17 cell differentiation. Apart from RORgammat, we found that AA147 decreases expression of the TCR-responsive factor IRF4, thus suppressing production of select effector cytokines across effector T cells, demonstrating additional ways in which this compound reshapes the activities of these essential T cell subsets. Our results demonstrate the potential for metabolically activated proteostasis regulators such as AA147 to selectively reshape TH17 cell identity while broadly dampening effector cytokine responses across effector T cell subsets, through both NRF2-dependent and independent mechanisms during T cell differentiation.

The premature aging disease Hutchinson-Gilford Progeria Syndrome (HGPS) results from the accumulation of progerin, a cytotoxic protein generated from a point mutation in the Lamin A/C gene, in the nuclear lamina. Upon the proper stimulation, cells degrade progerin, reversing cellular HGPS phenotypes; however, there is still a gap in our knowledge concerning which pathways are mediating progerin degradation. Previous data has demonstrated that the Anaphase Promoting Complex (APC), a multi-subunit ubiquitin ligase, tagets proteins for degradation, and that a decrease in APC function is linked with cellular aging. To determine if the APC is linked to HGPS disease phenotypes, we performed a meta-analysis of RNA-seq data from skin samples isolated from HGPS patients and identified dysregulation of several genes encoding subunits and substrates of the APC. Stimulation of APC activity decreased progerin protein levels and significantly decreased the number of cells with nuclear blebs. Proximity ligation assays (PLA) demonstrated that APC structure is compromised in HGPS cells and that APC stimulation increases proximity of the APC with progerin. Coimmunoprecipitation revealed that the APC co-activator, CDC20, physically interacted with nuclear lamina proteins. We further demonstrate that APC-mediated progerin degradation occurs through autophagy. Inhibition of the 26S proteasome enhanced progerin degradation, providing additional support for APC mediated-progerin degradation occurring independent of the proteasome. As such, we propose a previously unidentified interaction and mechanism by which cells remove progerin. This finding has impact on potential therapeutic strategies for HGPS, as well as providing further insight into linking the APC with both normal and premature aging.

Protein S-acylation by ZDHHC5 promotes membrane recruitment of the innate immune receptors NOD1 and NOD2. ZDHHC5 contains an extended cytosolic tail implicated in substrate recruitment, accessory-protein interactions, and enzyme trafficking, but its contribution to NOD1 and NOD2 modification remains unclear. Here, we used mutagenesis and complementation of ZDHHC5 knockout cells to define the regions required for NOD association, S-acylation, and plasma membrane localization. Substitution of three S-acylated cysteines in the tail preserved ZDHHC5 localization and its ability to restore NOD2 membrane recruitment. Progressive truncation revealed that most of the cytosolic tail was dispensable, with a construct comprising residues 1-230 retaining activity towards both NOD1 and NOD2. Further truncation to amino acids 1-218 impaired NOD S-acylation and membrane recruitment without eliminating NOD2 association. Thus, substrate association and productive modification have distinct requirements. C-terminal tail removal also differentially affected accessory-protein association: full-length ZDHHC5 co-immunoprecipitated with both GOLGA7A and GOLGA7B, whereas only GOLGA7B was detectably associated with the 1-230 construct. Together, these findings define a minimal functional ZDHHC5 that supports NOD S-acylation and distinguish the requirements for substrate association, catalytic activity, and accessory-protein interactions. They establish that the extended tail is dispensable for NOD modification under the conditions examined, while identifying differential requirements for GOLGA7A and GOLGA7B association with ZDHHC5.

Plants perceive and integrate diverse environmental signals through receptor kinase (RK) networks at the plasma membrane. Within this, Cysteine-Rich Receptor-Like Kinases (CRKs) constitute a large but not well-understood subfamily characterised by extracellular domains (ECDs) enriched in cysteine residues. CRKs have been implicated in plant responses to biotic and abiotic stress, as well as in developmental processes. Additionally, several CRKs have been proposed to act as redox sensors. Here, we investigate the homodimerization mechanism of Arabidopsis CRK18 and its regulation by redox conditions. By modulating pH and redox state, we assessed the stability and binding dynamics of the CRK18 ECD and its cysteine mutants. We also tested the plasma membrane localisation of all the cysteine mutants involved in the predicted disulfide bonds, and only CRK18C227A, C228A-ECD resembled the plasma membrane localization of wild-type CRK18. We combine co-immunoprecipitation, Forster resonance energy transfer-fluorescence lifetime imaging microscopy and microscale thermophoresis to quantify CRK18 self-association in planta and in vitro. Furthermore, we place CRK18 dimerization in a broader signalling context by identifying CRK18 interaction partners and CRK18-dependent signalling outputs. Using Arabidopsis CRK18 overexpression lines, we perform (phospho)proteomic and immunoprecipitation-mass spectrometry (IP-MS) analyses to map CRK18-centred signalling networks associated with stress-related responses. The constitutive activation of the CRK18 kinase domain and its interaction with many putative (cell wall) glycan-sensing RKs, including PERK15, suggest a regulatory role for CRK18. The absence of significant changes in the proteome and phosphoproteome of CRK18 overexpression lines in the absence of any trigger, and its restricted mobility after elicitation, suggest that CRK18 requires a stimulus for activation and possibly induces membrane microdomain reorganisation. This is in line with the infection assay with the nematode Heterodera schachtii, which causes modification and targeted damage to plant cell walls during infection, revealing that CRK18 acts as negative regulator of this process.

Cytos Multimodal Pooled Screening (CytosMPS) measures CRISPR guide identity, morphology, native 3' transcriptome, and protein abundance and localization in the same fixed cells. Following IL-1{beta} stimulation, cells with higher nuclear NF-{kappa}B show lower phospho-p38/HSP27 and higher NFKBIA/DUSP1 in the same cell, a measurement neither imaging nor transcriptomics alone provides. TGFBR2 and CTNNB1 perturbations produce distinct multimodal phenotypes. Guide representation and perturbation transcriptional responses agree with an orthogonal single-cell transcriptomic platform.

The timing of DNA replication and centrosome duplication is tightly regulated with cell cycle progression to ensure the faithful duplication of the genome during cell division. Both DNA and centrosomes are licensed for replication in late telophase/early G1, replicated in S phase and segregated during mitosis; yet how defects in DNA replication licensing are coupled to centrosome homeostasis remains poorly understood. Here, we show that depletion of the replication licensing inhibitor Geminin in proliferating mouse embryonic fibroblasts induces robust centrosome amplification together with impaired primary cilium assembly. Rather than promoting whole-genome reduplication, knockdown of Geminin triggers a replication stress response, characterized by DNA damage accumulation throughout the cycle, and activation of an ATR-dependent DNA damage response. Mechanistically, Geminin depletion-induced replication stress activates the ATR-Chk1-Wee1 checkpoint axis prolonging G2 and leading to premature centriole disengagement and centrosome amplification. These findings identify replication stress as the signaling module that couples defective DNA replication licensing to centrosome amplification.

Stress granules (SGs) are dynamic, membraneless cytoplasmic condensates that form in response to diverse cellular stressors. Although proposed to modulate stress responses by selectively sequestering proteins and RNAs, their precise molecular composition and function remain unclear. Reported SG transcriptomes differ substantially due to methodological discrepancies, notably between differential centrifugation (DC) and proximity labeling (PL). Here, we reanalyse publicly available human SG transcriptomes across multiple stressors, cell types, and isolation strategies. DC-based profiles were strongly shaped by RNA length, consistent with a physical bias inherent to the sedimentation-based separation. Correcting for this effect reveals limited concordance between studies. Mitochondrially encoded RNAs nonetheless consistently stand out as a distinctively regulated transcript class, without a uniform direction of enrichment/depletion across datasets, and with a modest but significant enrichment in our consensus SG signature. Immunofluorescence experiments further support, without definitively demonstrating, sequestration of mitochondrial dsRNA by SGs upon stress. Since leakage of mitochondrial nucleic acids is a well-characterised damage-associated molecular pattern linked to inflammation, their sequestration in SGs suggests a potential role in modulating immune-related stress responses. These findings refine our understanding of SG composition, underscore the need to control for technical artifacts in SG isolation, and provide a framework to distinguish genuine biological signals from methodological noise in SG research.

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

The budding yeast Saccharomyces cerevisiae is a well-established model organism to study cellular stress response and underlying mechanistic regulation. Although glucose starvation fundamentally alters gene regulation and cell behaviour, inconsistent deprivation protocols often trigger gross morphological artefacts. These non-specific changes confound findings by activating pathways independently of true glucose-signalling mechanisms. Furthermore, a thorough transcriptomic profile of glucose starvation using non-confounding conditions remains lacking. Consequently, the precise transcriptional impact of losing key metabolic regulators that mediate adaptation to glucose starvation remains undefined. Here we have employed a refined glucose starvation protocol, utilising raffinose exchange, which shows induction of vast transcriptional stress response with minimal impact on cellular morphology confirmed by label-free imaging. Transcriptomic profiling revealed shifts in metabolic regulation, ATP turnover, and cell-to-cell communication as acute glucose deprivation driving cells towards oxidation-driven metabolism. Additionally, we characterise transcriptional alterations seen in deletion mutants of SNF12 and SPT20, known regulators of cellular metabolism, showing previously unappreciated transcriptional conservation, in part mimicking glucose starvation response. Finally, we identified cargo and stress-specific expression related to both eisosome components and surface transporters that are critical for metabolic adaptation. Overall, this dataset provides a comprehensive transcriptomic resource for dissecting stress signalling and driving novel hypothesis generation.