Journal of Cellular Biochemistry

AO_SCPLOWBSTRACTC_SCPLOWThe cellular transcription factor BCL11b (B-cell CLL/lymphoma 11b) interacts with numerous cellular and viral factors to modulate gene expression positively or negatively. Post-translational modifications of BCL11b, such as SUMOylation and phosphorylation, have been documented to switch its transcriptional activity from a repressor to an activator state. In the present study, we investigated the acetylation of BCL11b and we identified the histone acetyltransferase p300 as able to acetylate BCL11b. Subsequently, we observed that the mutation of the lysine K686 residue of BCL11b (BCL11b K686R) influenced its global acetylation. Furthermore, the BCL11b K686R mutation also modulated the transcriptional regulation of BCL11b, including its activity in regulating the p21 and IL-2 promoters. This effect on transcriptional regulation was due to the importance of the lysine K686 residue for BCL11b nuclear localization. Our results underscore the critical role of the lysine K686 residue in BCL11b for its interaction with p300 and its nuclear localization, suggesting a possible function of p300 in the nuclear transport of BCL11b. Collectively, our findings contribute to a better understanding of BCL11b-mediated gene expression and of the interactions of BCL11b with cellular partners.

In yeast, transcriptional adaptor 2 (ADA2; SAGA complex subunit ADA2), a member of histone acetyltransferase (HAT) complex, regulates transcription through cell signalling, but its precise role in cellular metabolism remains unclear. In this study, genetic loss of ADA2 (ada2{Delta}) induces squalene (SQ) accumulation, indicating aberrant triterpene metabolism, coupled with endoplasmic reticulum (ER)/nuclear ER (nER) expansion. Lipid analyses of ada2{Delta} revealed elevated phosphatidic acid (PA) and phosphatidylcholine (PC) levels, indicating disrupted phospholipid metabolism. The expanded ER causes basal autophagy elevation, cellular recycling, and nER phagy, suggesting a regulatory role for ADA2 in autophagy. Downregulation of phosphatidate cytidylyltransferase (CDS1) and inositol-3-phosphate synthase (INO1), coupled with elevated PA and PC in ada2{Delta}, points to a significant disruption in cytidine-diphosphate-diacylglycerol and phosphatidylinositol pathway. Overexpression of CDS1 or INO1, or the inositol supplementation, in ada2{Delta} restores SQ, basal autophagy and ER phagy. The observed target of rapamycin Ser/Thr kinase complex (TORC1) activity in ada2{Delta} is due to the high PA content. Rapamycin-mediated inhibition of TORC1 reduced SQ, PA and ER expansion while increasing lipid droplets. In contrast, a rapamycin-treated ada2{Delta}pah1{Delta} strain retained high PA, SQ and ER expansion, underscoring the functional role of TORC1-nuclear envelope morphology protein 1 (Nem1)/sporulation-specific protein SPO7 (Spo7)-Pah1 axis. Notably, SQ levels remained unchanged in a rapamycin-treated ada2{Delta}atg39{Delta} strain, suggesting that loss of nER-phagy receptor, Atg39, impairs the effectiveness of TORC1 inhibition. In conclusion, our data unveiled a critical role for Ada2 in maintaining the intricate relationship between lipid and triterpene/sterol metabolism and connecting autophagy and ER homeostasis.

Autophagy contributes to normal cells physiology and is essential for progression of malignant tumors. While autophagy is mostly considered as a self-degradative and self-renewal process, it has non-degradative functions whose contribution to tumor progression is poorly explored. Here we use the autophagy dependent Drosophila RasV12, Scrib-/- carcinoma model to examine whether perturbation of distinct steps of autophagy differentially influences tumor progression. We found that inhibition of autophagosome formation, by mutating Atg13 or Atg6 either in the tumor or in the whole animal significantly decreased tumor growth. In contrast, blocking the later autophagosome-lysosome fusion (by loss of Vps39 or Syx17) and thereby autolysosomal degradation, does not reduce tumor size. We observed that an early (Atg13), but not a late (Vps39 or Syx17) block in autophagy showed reduced activity of JAK/STAT signaling, known to be critical for the progression of this tumor type. Importantly, we demonstrated that both Atg13 and Vps39 deficient tumors accumulated Stat92E inhibitor Su(var)2-10/dPIAS, a recently identified autophagic cargo, however in Vps39 mutants Su(var)2-10 is sequestered into autophagosomes. Finally, we found that reduction of Su(var)2-10 partially restores JAK/STAT signaling and rescues the growth of Atg13-deficient tumors, indicating its sequestration is a crucial mechanism to promote tumor progression.

Purine moieties are essential for many functions within the eukaryotic cell, including energy, signaling and nucleic acid synthesis. While purine starvation is known to induce stress resistance in eukaryotic model organism budding yeast Saccharomyces cerevisiae, it remains unclear whether the physiological response is related to disruption of synthesis pathway in particular position or it is uniform across all genetic deficiencies within the de novo adenine biosynthesis pathway. It is also not known how purine starved cells perceive purine shortage - weather they share the same signaling elements with nitrogen starvation or not. MethodsWe characterised physiology of strains with deletions in adenine biosynthesis pathway when cultivated in full or purine deficient and compared to cell physiological parameters when cultivated in nitrogen deficient media. We tested stress tolerance, carbon flux, cell cycle arrest and did transcription profiling (RNA-seq). ResultsOur findings demonstrate that purine starvation-induced stress resistance is significantly modulated by the specific step at which the pathway is interrupted. Transcriptional analysis revealed that purine starvation in many aspects phenocopies nitrogen starvation, particularly - in both starvations strong downregulation of ribosome related genes occurs. In the same time several metabolic features which differ from N- and ade- starvations: pentose phosphate pathway is specifically upregulated within ade4{Delta}-ade2{Delta} and downregulated in N-cells. Notably, the expression of stress-responsive genes such as HSP12, HSP26, and GRE1 varied between mutants, suggesting that the accumulation of pathway intermediates (e.g., AIR in ade2{Delta}) or the absence of downstream precursors (AICAR) alters the perception of starvation especially in the case of ade16{Delta}ade17{Delta} strain. ConclusionsMetabolic and stress-tolerance phenotypes of purine auxotrophs are not merely a result of purine depletion but seems that the response is signalled via the same pathways, like TOR1. The results suggest that strains having mutations within various positions of the purine pathway "perceive" purine limitation a bit differently - especially when we compare the end of the pathway with the other mutants. Different phenotypic outcomes of the occasional purine depletion might give preferences for organisms which have mutations in the beginning rather at the end of the pathway. Besides, our findings might have implications in the design of synthetic pathways and the use of auxotrophic markers in yeast research.

MRPL47 (Mitochondrial Ribosomal Protein Large Subunit 47) gene in chromosome 3q26 encodes a protein that is part of the large subunit of the mitochondrial ribosome. We observed that MRPL47 is frequently amplified and overexpressed in ovarian cancer samples. Importantly, increased expression of MRPL47 mRNA is associated with high levels of MRPL47 protein in ovarian cancer patients. High expression of MRPL47 is also associated with poor overall and recurrence free survival of ovarian cancer patients. Notably, MRPL47 improved metabolic fitness by enhancing cellular respiration, and glycolysis in cancer cells. Gene set enrichment analysis and target specific knockdown assays revealed that MYC transcription factor regulates MRPL47 expression. Furthermore, MRPL47 was identified very high in the plasma samples of ovarian cancer patients compared to those of healthy volunteers. MRPL47 was also associated with cisplatin resistance, whereas its expression predicted sensitivity to cisplatin therapy. Taken together, we demonstrated that MRPL47 can be used as a diagnostic biomarker for ovarian cancer and other cancers with 3q26 chromosomal amplification.

Translation is a crucial regulatory mechanism involved in several diseases, including cancer, where pro-inflammatory conditions within the microenvironment have been shown to modulate the translation of specific mRNAs. In the present study, we focused on the regulation of insulin growth factor-like family member 1 (IGFL1) in MCF7 breast cancer cells in response to pro-inflammatory IL-1{beta} and observed an induction of both transcription and translation. We characterized the 3 untranslated region as regulatory hub for the post-transcriptional regulation and identified a distinct G-rich region to confer the IL-1{beta}-dependent translational increase. Our study therefore provides new insights into the translation regulation of IGFL1 in the context of an inflammatory tumor microenvironment.

SignificanceQuantifying lipid droplet (LD) remodeling in 3D hepatic organoids is often limited to endpoint staining or phototoxic live fluorescence imaging, thereby obscuring droplet-level kinetics. AimWe aimed to develop a label-free method to track LD dynamics in living hepatic organoids under different fatty-acid loads. ApproachTime-lapse 3D refractive-index tomograms were acquired using holotomography and analyzed with a depth-adaptive, multi-threshold segmentation pipeline to quantify LD number, volume, sphericity, and refractive-index-derived concentration and dry mass at single-droplet resolution. ResultsOleic acid and linoleic acid induced LD accumulation while preserving organoid integrity, whereas palmitic acid triggered rapid structural collapse. Despite increases in total LD burden under both oleic acid and linoleic acid, droplet-level dynamics diverged: oleic acid produced volume-dominated accumulation via enlargement of fewer LDs and increased size heterogeneity, whereas linoleic acid produced number-dominated accumulation via sustained increases in LD number, yielding a more uniform population of small droplets. ConclusionsLabel-free holotomography with depth-adaptive analysis enables non-invasive, longitudinal, and multi-scale quantification of LD dynamics in intact organoids and reveals fatty-acid- dependent temporal modes of lipid storage. Statement of DiscoveryWe developed a label-free, longitudinal 3D holotomography framework with depth-adaptive lipid droplet segmentation that quantifies single-droplet dynamics in living mouse hepatic organoids. Using this platform, we found that oleic acid and linoleic acid induce LD accumulation via distinct strategies--oleic acid via droplet enlargement and linoleic acid via sustained increases in droplet number--while palmitic acid rapidly compromises organoid integrity.

BackgroundIdiopathic epilepsy (IE) is the most common chronic nervous system disorder of dogs, and its cause is poorly understood. Emerging evidence suggests that microbiome alterations can occur with IE via the microbiota-gut-brain axis. Therefore, we analyzed the fecal microbiomes of 98 dogs (49 IE, 49 control) in a pairwise case-control observational study using 16S rRNA gene sequencing. ResultsAlthough the microbial community was mostly similar between groups, IE was associated with a modest but significant shift in Weighted-Unifrac distance (P = 0.042). We used six differential abundance (DA) methods to identify differentially abundant amplicon sequencing variants (ASVs) between IE and control groups. Notably, one Collinsella ASV was found to be significantly more abundant in IE dogs by all six methods. The gut microbial compositions varied drastically across households (accounting for about 69% of the total variation), but did not have significant differences between sex, age, or breed. Phenobarbital administration in IE dogs had a significant effect on seizure control, and was not associated with changes in the microbiome. ConclusionOur findings suggest a relationship between gut microbiomes and IE. However, the specific mechanism needs to be further investigated.

Osteosarcoma is the most common pediatric bone tumor yet has limited treatment options, especially for metastatic cases with a 20% adjusted 5-year survival rate. Current therapies are non-specific, involving primary tumor resection with DNA-damaging chemotherapies like methotrexate, doxorubicin, and cisplatin. Few effective treatment options exist for metastases. Targeting metabolism involving cancers reduced mitochondrial functionality remains underexplored in osteosarcoma. We investigated the therapeutic potential in human osteosarcoma primary and metastatic cell lines of metabolic modulating drugs including metformin, cycloheximide, mitochondrial ETC inhibitors (antimycin A, metformin), dichloroacetate, and imipridones (ONC201, ONC206) on mitochondrial function and cell viability, individually and combined under various nutrient conditions across our lines. Results confirmed osteosarcoma cells are more dependent on glucose than osteoblasts but also require mitochondrial function for survival, highlighting the therapeutic potential of metabolic pathways. Osteosarcoma cell viability was reduced when any metabolic drug treatment was combined with conditions forcing reliance on mitochondrial OXPHOS capacity. Combination metabolic therapies, particularly ONC201/ONC206/metformin in 143B cells, and to a lesser extent DCA and ONC201 with either ONC206 or antimycin A, showed enhanced cytotoxicity compared to single agents, with a good therapeutic index based on minimal toxicity to normal osteoblast cells. The degree of effectiveness varied across cell lines, underscoring the importance of personalized treatment strategies. RNA-Seq transcriptome analysis revealed that effective nutrient and metabolic drug treatments triggered widespread regulatory changes in osteosarcoma cells involving increased translation/splicing with decreased mitochondrial processes such as cholesterol biosynthesis. These results demonstrate the utility of developing combined metabolic and chemotherapeutic treatments for osteosarcoma.

Tuberous sclerosis complex (TSC) is an autosomal dominant disorder caused by mutations in the TSC1 and TSC2 genes and is characterized by benign hamartoma formation in multiple organs. The TSC1-TSC2 complex regulates mTORC1 signaling in response to cellular growth conditions. This study aims to predict the structural stability and functional effects of non-synonymous single-nucleotide polymorphisms (nsSNPs) in human TSC1 and TSC2 using computational approaches. Twelve computational tools were assessed using receiver operating characteristic (ROC) analysis and applied to identify deleterious nsSNPs. Protein stability was predicted using I-Mutant 2.0 and MUpro, while evolutionary conservation was analyzed with ConSurf. NetPhos 3.1 identified potential PTM sites, and MutPred2.0 evaluated their functional impact. Project HOPE assessed mutation-induced physicochemical changes. Structural models were validated using multiple tools, visualized in ChimeraX 1.9, and further evaluated by molecular dynamics simulation to confirm wild-type and mutant stability. All twelve tools had AUC values above 0.90. A combined in silico analysis identified twelve high-risk nsSNPs in TSC1 and sixteen in TSC2, all reducing protein stability, located in conserved regions, and potentially disrupting phosphorylation sites. MutPred and Project HOPE confirmed their impact on protein function. Functional analysis showed TSC1 and TSC2 affect mTORC1 and PI3K-Akt pathways. RMSF and RMSD analyses revealed that TSC1 variants rs1846545280 (G236E), and rs2132135678 (V234E), and TSC2 variants rs45517223 (S758C), rs2151354925 (T836P), and rs45517365 (R1570W) had the largest structural fluctuations. Substitution with glutamic acid, a negatively charged and bulkier residue, may disrupt local folding of TSC1. Similarly, replacement of arginine with tyrosine at position 1570 may impair Rheb binding at the GAP domain of TSC2. These findings highlight potentially pathogenic nsSNPs in TSC1 and TSC2.

PurposePancreatic ductal adenocarcinoma (PDAC) is characterized by a nutrient-deprived and hypoxic tumor microenvironment (TME) that imposes severe metabolic stress on cancer cells. Under these conditions, tumor cells frequently activate the integrated stress response (ISR) to adapt to TME and develop resistance to therapies. However, how TME components support tumor adaptation to mitochondrial metabolic stress remains incompletely understood. Here, we aimed to identify key metabolite involved in ISR adaptation under oxidative phosphorylation (OXPHOS) inhibition and to elucidate the metabolic symbiosis between cancer-associated fibroblasts (CAFs) and PDAC cells. MethodsWe integrated transcriptomic and metabolomic analyses with functional assays. ISR activation was evaluated by assessing the phosphorylation of eIF2 (p-eIF2) following treatment with carboxyamidotriazole orotate (CTO), an Complex I inhibitor. Metabolomic profiling was used to identify metabolites involved in ISR activation alleviation. Mouse models were used to assess therapeutic responses following depletion of the identified metabolite under CTO treatment. Genetic perturbation of Slc38a4 was performed to assess its functional role in tumor cell adaptation to metabolic stress. ResultsWe identified asparagine (ASN) as a critical metabolite supplied by CAFs to PDAC cells under OXPHOS inhibition. A minimum level of ASN is required for PDAC cells to execute ISR downstream adaptation. ASN depletion significantly enhanced the anti-tumor efficacy of OXPHOS inhibition both in vitro and in vivo. SLC38A4 emerged as a potential mediator of this interaction. SLC38A4 expression was associated with c-Myc, and its loss increased the sensitivity of PDAC cells to CTO-induced metabolic stress. ConclusionOur findings reveal a CAF-tumor metabolic crosstalk in which stromal-derived ASN supports PDAC cell adaptation to mitochondrial metabolic stress. Adaptive outcome of ISR signaling depends on the availability of key metabolic substrates such as ASN. When extracellular ASN supply is limited, the ATF4-dependent adaptive program collapses, converting ISR from a pro-survival response into a therapeutic vulnerability. SLC38A4 may function as a key mediator of this metabolic coupling and represents a potential target for enhancing the efficacy of OXPHOS inhibition in PDAC.

The PI3K-AKT-MTOR signalling axis is pivotal in regulating cell survival, proliferation, and growth. TSC2 (tuberous sclerosis complex subunit 2) is a well-established negative regulator of this pathway, which primarily acts by suppressing the MTORC1 activity. While the cytoplasmic role of TSC2 is well characterized, emerging evidence suggests its additional nuclear functions. Previous work from our laboratory identified TSC2 as a transcriptional repressor of the EREG (Epiregulin) gene. Building on this foundation, the present study investigates the transcriptional role of TSC2 in miRNA (microRNA) gene regulation. A genome-wide miRNA microarray profiling of TSC2-depleted cells from an oral squamous cell carcinoma (OSCC) cell line, SCC131, identified 19 upregulated and 24 downregulated miRNAs. Of them, miR-514b-3p emerged as one of the most significantly upregulated miRNAs. TSC2 knockdown resulted in robust miR-514b-3p upregulation, whereas TSC2 overexpression suppressed its expression. Moreover, TSC2 negatively regulates MIR514B promoter activity in an NLS-dependent manner. The chromatin immunoprecipitation analysis showed direct binding between TSC2 and MIR514B promoter, establishing miR-514b-3p as a transcriptional target of TSC2. We further identified TSPAN9 (Tetraspanin 9) as a direct downstream target of miR-514b-3p. The dual-luciferase reporter assay and Western blot analysis confirmed direct interaction between miR-514b-3p and TSPAN9 3UTR. Furthermore, TSC2 positively regulates TSPAN9 levels by repressing miR-514b-3p, thereby establishing a novel TSC2-miR-514b-3p-TSPAN9 regulatory axis. Additionally, we uncovered crosstalk between TSC2-miR-514b-3p-TSPAN9 axis and the canonical PI3K-AKT-MTOR signalling, where miR-514b-3p positively, and TSPAN9 negatively regulates the PI3K-AKT-MTOR pathway. Interestingly, AKT functions as an upstream regulator of this axis by modulating TSC2 nuclear localization. Collectively, this study provides new insights into the non-canonical, nucleus-dependent transcriptional functions of TSC2, thus expanding its role beyond cytoplasmic signalling regulation and underscoring its significance in the cellular signalling networks.

Epilepsy is one of the most prevalent neurological diseases, with 25-33% of patients developing drug-resistant epilepsy (DRE). The precise etiology of DRE remains unidentified. Recent studies have revealed an increase in tetraploid astrocytes in drug-resistant temporal lobe epilepsy (DR-TLE), a common subtype of DRE. This study aims to characterize the function of tetraploid astrocytes in the brain of subjects without central nervous system diseases and in DR-TLE. Cortical samples adjacent to the epileptogenic zone were obtained from DR-TLE patients undergoing resective neurosurgery and from postmortem donors without neurodegenerative, neurological, or psychiatric disorders. Tetraploid astrocytes were identified using the astrocytic marker NDRG2, and their functional characterization was assessed by evaluating markers of metabolism (ALDH1L1), transport (SOX9), electric function (NF1A), or reactive astrocytes (NF{kappa}B p65 and pSTAT3), via immunostaining followed by flow cytometry. Tetraploid astrocytes expressed all functional markers tested. The percentage of tetraploid astrocytes expressing ALDH1L1 or SOX9 was significantly increased in DR-TLE with respect to controls, whereas NF1A remained unchanged. Inflammatory markers pSTAT3 and NF{kappa}B p65 showed an upward trend in 4C astrocytes. In contrast, diploid (2C) astrocytes expressing these markers were reduced in DR-TLE, suggesting a functional shift toward polyploid cells in the DR-TLE cortex. Our findings suggest the preservation of markers of metabolism, transport and electric function in tetraploid astrocytes in physiological conditions and in DR-TLE patients. Moreover, the astrocytes with metabolic and transporter markers were significantly increased in DR-TLE. These findings point to tetraploid astrocytes as potential contributors to DR-TLE mechanisms.

Parkinsons disease (PD) is the second most common neurodegenerative disease, resulting from accumulation of -synuclein (-syn) in midbrain dopaminergic neurons and progressive neuronal loss. The most relevant species of -syn, oligomers, may exert neurotoxicity in a variety of mechanisms. Accumulation of misfolded -syn in the endoplasmic reticulum (ER) lumen induces ER stress conditions that leads to activation of the Unfolded Protein Response (UPR) and its main sensor PKR-like ER kinase (PERK). PERK is critical for cell fate determination - under prolonged ER stress, it may direct cell towards pro-apoptotic pathways. Targeting of -syn aggregation or UPR by genetic and pharmacological approaches proved effective in preclinical models of PD by previous research. Thus, in the present study, we aimed to determine the potential effect of combination of small-molecule inhibitors of -syn aggregation and ER stress-mediated PERK signaling (namely anle138b and AMG44) in a novel, 3D in vitro model of PD. We demonstrate that combination of both anti-aggregation and ER stress-targeting approaches amplifies neuroprotection against PD in organoid model in terms of increased neuronal metabolic activity, decreased -syn phosphorylation and aggregation, reduced dopaminergic cell death, and restoration of proteostasis.

The equilibrium between cell death and cell division is crucial for maintaining tissue homeostasis in a multicellular organism. Apoptosis plays an essential role in preserving homeostasis and hence occurs in a coordinated manner. However, inhibition of apoptosis is one of the hallmarks of cancer. Apoptosis Inhibitor 5 (Api5), an anti-apoptotic protein, is upregulated in various cancers, including ovarian, bladder, cervical, and lung cancers. Studies have demonstrated that altered expression of Api5 leads to the transformation of non-tumorigenic breast epithelial cells. However, the mechanism regulating this process is not well-elucidated. Our study demonstrates that overexpression of Api5 increased FGF2 (Fibroblast Growth Factor 2) levels both at protein and transcript levels. We studied the mechanistic details of changes in morphology, proliferation, and polarity observed upon FGF2/FGFR1 deregulation in Api5-overexpressing cells. Deciphering the signalling mechanism underlying Api5-FGF2-mediated breast tumorigenesis revealed that the PDK1/Akt and Ras/MAPK/ERK pathways regulated multiple transformation phenotypes. PDK1/Akt enhanced proliferation and altered morphology during initial stages, whereas Ras/MAPK/ERK regulated polarity disruption, proliferation, and reduced apoptosis during later stages of morphogenesis. In conclusion, this study provides insights into the signalling mechanism regulating the transformation phenotypes associated with Api5 overexpression in a non-tumorigenic breast epithelial cell line.

Modification by ubiquitination governs the half-lives of thousands of proteins that are fated for elimination by either the proteasome or autophagy pathways, depending on the intricate architectures of ubiquitin modification. This system mediates quality control for individual proteins, protein complexes, and organelles, as well as myriad purely regulatory functions. Here we provide a comprehensive survey of the ubiquitin-proteasome system (UPS), the scope of which is at present poorly defined. The UPS, with the inclusion of pathways involving ubiquitin-like modifiers, comprises in our estimate over 1400 distinct proteins in humans, a vast set of activities whose collective impact on the biology of the cell is pervasive. The UPS is an integral component of the proteostasis network (PN), the remainder of which we have also surveyed in recent studies. With the addition of molecular chaperones, proteins from autophagy-lysosome pathway, and related activities, the PN includes in total over 3100 components by our estimates. Comprehensive and systematic definition of these pathways should support a range of ongoing investigations in the areas of genomics, proteomics, biochemistry, cell biology, and disease research.

Colorectal cancer is the third most common cancer across the world. Acquired resistance to therapeutics is one of the major challenges in cancer cure. With the development of resistance to organometallic drugs there was switch for the usage of inhibitors of kinases which play a crucial role in cellular activities. Regorafenib is a multi-kinase inhibitor used as an oral anti-cancer drug for treating advanced colorectal cancer (CRC). With increasing reports of acquired resistance to regorafenib in long-term use it is inevitable to understand the mechanisms underlying in the development of resistance. To understand the molecular mechanism of acquired drug resistance towards regorafenib we have developed a regorafenib resistant HCT116 cell line (Reg-R-HCT116) and an integrated quantitative proteomic and phospho-proteomics approach is used to elucidate the molecular signaling mechanisms that help in drug tolerance. Proteome and Phosphoproteome analysis revealed an extensive remodelling of signaling pathways associated with metabolism, protein synthesis and stress adaptations. This also revealed a large set of phosphorylated proteins as well as proteins that might be associated with aberrant activation or differential alteration of PI3K-AKT-mTOR, EIF2, HIF-1, Apoptosis inhibition, Glucose metabolism, amino acid metabolism and DNA-repair-associated signaling. Differential phosphorylation of downstream molecules of the mentioned pathways NDRG1, ACINUS and RICTOR and enhanced cell survival further confirmed their role in drug tolerance. Targeted inhibition of both the mTOR complexes using Torin1 resensitized the cells to regorafenib. Combination treatment of regorafenib with torin1 showed a synergistic cytotoxicity and attenuated the expression of key survival proteins. These findings provide mechanistic insight into acquired resistance to regorafenib in colorectal cancer and identified mTOR/eIF2 signaling as one of the critical drivers of resistance phenotype. The results suggest combinatorial targeting of these pathways could be an effective strategy to overcome regorafenib resistance and improve clinical outcomes in CRC.

The MYC associated factor X (MAX) is the heterodimeric partner of the MYC paralogs (MYC, MYCN and MYCL). When deregulated, high level of the MYC paralogs contribute to all aspects of tumorigenesis and tumor growth. MAX can also heterodimerize with the MXD proteins, MNT and MGA. Heterodimerization and sequence specific DNA binding to the E-Box sequences at gene promoters is controlled by their heterodimerization with the MAX b-HLH-LZ. As a heterodimer with MAX, MYC proteins activate genes involved in cell metabolism, growth and proliferation whereas MXD proteins, MNT and MGA repress them. MAX can also bind to the E-Bos sequence as a homodimer. Being devoid of a transactivation domain it can act as an antagonist of the MYC/MAX heterodimers. Variants of MAX have been reported to be linked to cancer. These variants are either not expressed, inactivated or lead to missense mutations. This has led to the notion that MAX may have a tumor suppressor role. Here, we characterize three of those variants with missense mutations in the basic region, i.e. E32K, R35P and R35C. We analyzed their heterodimerization with the b-HLH-LZ of MYC and their DNA binding properties as homo-and heterodimers. The R35C variant b-HLH-LZ was found to have a markedly increased affinity for the b-HLH-LZ of MYC. We also observed that all three b-HLH-LZ variants have a lower affinity as homodimers for the E-Box than the WT. This was shown to lead to a preferential binding of all the heterodimeric b-LHLH-LZ to the E-Box. This effect is exacerbated in the case of the R35C variant. We argue that this preferential binding of MYC as heterodimers with these variants to E-Box sequences could contribute to tumorigenesis. Hence, our results suggest that, mechanistically, the MAX homodimer bound to the E-Box could act as a tumor suppressor. MATERIALS AND METHODSO_ST_ABSMolecular modelingC_ST_ABSThe open source version 1.7.6.0 of Pymol was used for modeling and molecular rendering [1]. The crystal structure of the MAX homodimer bound to the E-Box (1HLO [2]) was used as a template for the generation of the models. The variants were generated using the mutagenesis function in the wizard. The conformation of the K32 side chain was manually set in order to avoid introducing steric clashes with DNA. Protein expression and purificationThe cDNA, coding for the MAX b-HLH-LZ (Max* hereafter, residues 22-103, UniProt entry P61244-1) to which are added the GSGC residues in c-terminal, inserted in the pET3a vector was already available in the laboratory [3] and was used as a template to generate the plasmids with inserts coding for each of the mutants (E32K, R35C and R35P) through quick-change PCR with Q5 DNA polymerase and DpnI from New England Biolabs. The primers used were purchased from IDT DNA, their sequences are listed in Table S1. Sequence for each construct was confirmed by Sanger sequencing at the Plateforme de sequencage SANGER - Centre de recherche du CHU de Quebec - Universite Laval. The primary structure for the basic region of each construct is given in Fig. 2A. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=137 SRC="FIGDIR/small/715400v1_fig2.gif" ALT="Figure 2"> View larger version (41K): org.highwire.dtl.DTLVardef@1b05d5eorg.highwire.dtl.DTLVardef@1c1d692org.highwire.dtl.DTLVardef@ee469dorg.highwire.dtl.DTLVardef@15e0ba4_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFigure 2.C_FLOATNO Structure schematics, specific and non-specific interactions dictating specificity and stability of binding of the basic region of MAX to the canonical (CACGTG) E-Box. A. Primary structure for the basic region of MAX and each of the variants. Positions making the most important contacts with the E-box are indicated by black arrows. Positions for the variants studied here are colored according to the Zappo colour scheme, following their physico-chemical properties: red for negative, blue for positive, magenta for proline and yellow for cysteine. B. The side chain (carboxylate) of E32 receives H-Bonds from the CA nucleobases in the leading strand (white carbon atoms). R35 and R36 make a salt bridges with phosphate groups while and the guanidino moiety of R36 makes a specific H-Bond with the nucleobase of the G in the strand of the reverse complement (cyan carbon atoms). C. The R35C mutation removes one non-specific salt-bridge at the interface of the complex. D. The aliphatic portion of the K side chain in the E32K variant is unable to accept the H-Bonds from the CA nucleobases and leads to the stabilisation of the complex and the helical structure of the basic region. E. In addition to removing a salt-bride, the Pro residue in the R35P kinks the path of the basic region, prevents the establishment of the specific H-Bonds mandatory for recognition of the E-Box and leads to unfolding of the helical state. C_FIG The MYC b-HLH-LZ (Myc*), the Max*WT b-HLH-LZ and its variants were expressed and purified as previously described [3,4] After lyophilisation, the b-HLH-LZs were kept at -20{degrees}C and solubilised in Myc buffer (50 mM NaCl, 50 mM NaH2PO4 pH 5.5) for Myc* or PBS for Max* at a final concentration of 1 mM before use. Circular dichroismAll circular dichroism (CD) measurements were performed on a Jasco J-810 spectropolarimeter equipped with a Peltier-type thermostat. The instrument was routinely calibrated using an aqueous solution of d-10-(+)-camphorsulfonic acid at 290.5 nm. Samples were prepared as follows: Max* (either WT or a variant) was diluted in 100 {micro}l 2X CD buffer (40 mM KCl, 11.4 mM K2HPO4, 28.6 mM KH2PO4, pH 6.8) and the volume adjusted to 106 {micro}l with PBS. 10 {micro}l TCEP 16 mM were added, and the volume further adjusted to 192 {micro}l with ddH2O before samples were incubated overnight at room temperature. After reduction, Myc* was added and the volume adjusted to 198 {micro}l with Myc buffer (Na2HPO4 0.95 mM, NaH2PO4 49.05 mM, 50 mM NaCl, pH 5.5). The DNA complexes were prepared as follows. After a 10 minutes incubation of the protein samples at room temperature, 0, 1 or 2 {micro}l of 2 mM of specific or non-specific DNA duplexes in 10 mM Tris pH 8.0 were added and the volume adjusted to 200 {micro}l with 10 mM Tris pH 8.0. The strands of the specific probe were: 5-ATT ACC CAC GTG TCC T*AC-3 and 5-GTA GGA CAC GTG GGT* AAT-3 (with the E-box sequence underlined) and the non-specific probe: 5-ATT ACC TCC GGA TCC T*AC-3 and 5-GTA GGA TCC GGA GGT* AAT-3 (Integrated DNA Technologies). Samples were further incubated for 10 minutes at room temperature and transferred to a 1 mm path length quartz cuvette. All spectra were recorded from 250 to 195 nm at 0.1 nm intervals by accumulating 10 spectra at 25 {degrees}C. Thermal denaturations were recorded at 222 nm from 5 to 95 {degrees}C at a heating rate of 1 {degrees}C/min. CD signal for spectra and thermal denaturations was corrected by substracting the signal from corresponding spectra or thermal denaturation either for buffer alone or the appropriate DNA duplex. CD signal was then converted to mean residue ellipticity using the following formula [5]: [{theta}] = {delta} {middle dot} MRW/(10{middle dot}c l) where [{theta}] is the mean residue ellipticity in deg {middle dot} cm2 dmol-1, {delta} is the CD signal in millidegrees, MRW is the mean residue weight, c is the concentration in mg/ml and l is the pathlength in mm. For the heterodimers, the concentration used was the sum of Max* and Myc* and the MRW was determined using a weighted average.

The enzyme nitric oxide synthase (NOS) breaks down the semi-essential amino acid L-arginine (L-Arg) in the cell to produce citrulline and nitric oxide (NO). NO is a crucial signalling molecule in cells that controls the metabolism of fats and carbohydrates. The aim of this study was to investigate two important genes in the L-Arg-NOS-NO signalling pathway, AMPK and ACC-1, as markers of the molecular mechanisms that are triggered when liver cells sense elevated L-Arg. Mouse liver epithelial insulin-sensitive BNL CL2 cells were used as a model system and cultured with 0, 400 or 800 {micro}M L-Arg. Cell growth parameters were analysed alongside qRT-PCR based analysis of target transcripts involved in lipid and glucose metabolic pathways. In a further experiment, NOS inhibitor; L-NAME (40 mM) and external NO donor; SNAP (100 {micro}M) were added and the effect on target gene expression analysed. L-Arg addition impacted culture viability and cell growth. AMP-activated protein kinase (AMPK) was regulated in response to L-Arg addition with increasing extracellular concentrations elevating AMPK mRNA and protein expressions. L-NAME decreased target gene expression in an L-Arg addition dependent manner. SNAP (100 {micro}M) addition increased target gene expression after 6 and 24 h. NO, produced as a result of L-Arg addition and the factors L-NAME and SNAP, that regulate NO bioavailability, impacted BNL CL2 cell NO/AMPK/ACC-1 signalling pathways via regulating mRNA expression and subsequently protein expression.

The transcription factor Pho4 is crucial for the response to phosphate starvation in many fungi, and it has been linked to tolerance to alkalinization of the medium and to pathogenicity. It is widely accepted that it is encoded by a single gene. However, the industrially relevant yeast Komagataella phaffii might contain two Pho4-encoding genes (PAS_chr1-1_0265 and PAS_chr2-1_0177, designated here PHO4(A) and PHO4(B), respectively), which have never been functionally characterized. The phenotypic analysis of single and double mutants suggests that Pho4(B) plays a major role in the adaptation to Pi scarcity. While single mutants exhibited limited and non-overlapping phenotypic defects, the pho4(A) pho4(B) strain was sensitive to multiple types of stress, including phosphate starvation and alkaline pH. Transcriptomic analysis confirms that Pho4(B) is crucial for the transcriptional response to phosphate starvation, including induction of typical gene markers (PHO5, PHO89, VTC1, etc.). However, by using a GFP reporter we found that PHO4(A) also participates in the induction of PHO89 under high pH stress. Expression of both PHO4(A) and PHO4(B) in S. cerevisiae complemented the pho4 mutation under phosphate limitation by restoring growth, expression of the Pho84 transporter and secreted phosphatase activity. These results indicate that both transcription factors display partially overlapping functions, responding differently to diverse stimuli, and that together they constitute a key component in the adaptation to a variety of stresses. Therefore, K. phaffii is an exceptional example among fungi that encodes two Pho4 functional transcription factors.