Search Results (1,630)

Mitochondrial dysfunction in colonic smooth muscle cells (SMCs) is closely associated with impaired gut motility in functional constipation (FC), but the underlying molecular mechanisms remain incompletely understood. The mitochondrial unfolded protein response (UPR^mt) is a critical pathway for maintaining mitochondrial proteostasis, and heat shock factor 1 (HSF1) acts as an important upstream regulator of this response. In the present study, we employed a loperamide-induced FC mouse model, combined with single-cell transcriptomic, molecular, and functional analyses to characterize the HSF1-UPR^mt pathway in colonic SMCs and to investigate its role in FC. Single-cell transcriptomic analysis of colon tissue from FC mice revealed marked downregulation of UPR^mt-associated genes in colonic SMCs. Immunofluorescence, Western blotting, and RT-qPCR analyses of colonic tissue confirmed that HSF1 expression was reduced in colonic SMCs, along with the downregulation of the UPR^mt components, including HSP60, mtHSP70, and LONP1. These molecular changes were accompanied by mitochondrial structural damage, seen by transmission electron microscopy, and by functional impairments, including reduced mitochondrial membrane potential, elevated mtROS production, decreased ATP levels, and diminished activities of respiratory chain complexes I–V. AAV9-mediated overexpression of HSF1 reactivated the UPR^mt pathway, improved mitochondrial function, and ameliorated constipation, whereas shRNA-mediated knockdown of HSF1 further suppressed UPR^mt activity and aggravated mitochondrial damage, indicating that HSF1 bidirectionally regulates this pathway. Complementary experiments in primary colonic SMCs confirmed that this regulatory mechanism operates in a cell-autonomous manner, as modulation of HSF1 expression produced corresponding changes in the UPR^mt pathway, in the expression of mitochondrial respiratory chain complex subunits (ATP5A, NDUFA9, COX1, SDHA, UQCRC1), and in ATP production, mirroring the in vivo findings. Collectively, these results demonstrate that HSF1 plays a pivotal role in maintaining mitochondrial homeostasis in colonic SMCs through regulation of the UPR^mt pathway and that HSF1 dysfunction is closely associated with slowed gut motility in FC. These findings offer a new mechanistic perspective on FC and point to the HSF1–UPR^mt axis as a potential therapeutic target. Full article

Azole resistance in Candida albicans is an increasing clinical challenge. Upc2 is a key transcription factor regulating ergosterol biosynthesis, but its additional roles in azole tolerance remain unclear. This study investigated whether Upc2 contributes to azole resistance through pathways beyond ergosterol synthesis. Chemical sensitivity screening, RNA sequencing, flow cytometry, and molecular assays were performed to compare wild-type C. albicans and the upc2Δ/upc2Δ mutant under fluconazole (FLC) treatment. The UPC2 gene deletion affected physiological processes that are dependent on the calcineurin signaling pathway and led to an overall negative enrichment trend in the unfolded protein response (UPR) pathway gene set. Mechanistically, the UPC2 gene deletion impaired unconventional splicing of HAC1 mRNA, leading to accumulation of unfolded proteins and phenotypically its deletion enhanced sensitivity of C. albicans to FLC in planktonic growth, hyphal development, and biofilm formation. Our findings reveal that Upc2 regulates proteostasis in C. albicans, and its absence enhances FLC efficacy by disrupting the UPR pathway. Targeting Upc2-mediated UPR signaling may represent a promising strategy to combat azole resistance. Full article

Protein kinases have key roles in cells as they regulate diverse signal transduction pathways. Mitogen-activated protein kinase (MAPK) signaling route modulates several processes, such as cell proliferation, cell programming, metabolic changes and stress responses. Within the group of proteins participating in this pathway, the MAPK kinase (MEK1) is a dimeric, 393-residue-long, dual-specificity protein kinase that phosphorylates both tyrosine and threonine residues. In this study, we explored the conformational changes occurring during the unfolding of MEK1, by using orthogonal biophysical techniques. Intrinsic fluorescence, extrinsic 8-anilinonapthalene-1-sulfonic acid (ANS) fluorescence, dynamic light scattering (DLS), and far-ultraviolet (UV) circular dichroism (CD) showed that the protein acquired a native-like conformation within a narrow pH range (8.0 to 9.0). Urea and guanidinium hydrochloride (GdmCl) denaturations followed by intrinsic and ANS fluorescence and far-UV CD, at pH 8.1, where the protein acquired a native-like conformation, showed that: (i) the apparent conformational stability of isolated MEK1 was low; and (ii) the unfolding occurred through the presence of intermediates. The presence of several unfolding intermediates was also evidenced through: (i) differential scanning calorimetry (DSC) in the absence of the ligand ATP; and (ii) unfolding simulations with the help of computational techniques based on constraint network analysis (CNA). We propose that the apparent low stability of this protein was related to its flexibility and modulates its ability to interact with diverse molecular partners. Full article

The kidney is a highly specialized organ that maintains systemic homeostasis through tightly coordinated cellular and molecular mechanisms. Renal parenchymal cells regulate metabolic waste excretion, electrolyte and acid–base balance, and blood pressure control—functions that rely on the dynamic integration of intracellular organelles. Recent advances in molecular and biochemical research have highlighted how inter-organelle communication is essential for preserving renal cell function and adaptive responses to stress. This review focuses on the molecular crosstalk among key organelles—including the nucleus, endoplasmic reticulum (ER), Golgi apparatus, mitochondria, lysosomes, and peroxisomes—primarily in tubular epithelial cells. We discuss how these interactions coordinate metabolic signaling, protein homeostasis, redox balance, and energy production and how their disruption contributes to maladaptive pathways during acute kidney injury (AKI), ultimately promoting chronic kidney disease (CKD) transition. Particular focus is placed on emerging pathways linking organelle dysfunction to inflammation, fibrosis, and metabolic reprogramming. Furthermore, we highlight recent advances in genetics and molecular therapeutics targeting organelle communication, including modulation of ER stress responses, mitochondrial biogenesis, and lysosomal function. Clinically approved agents, such as mTOR inhibitors, and experimental approaches—such as chemical chaperones and mitochondrial transplantation—demonstrate the potential to restore organelle homeostasis and mitigate renal injury. Overall, elucidating the molecular networks governing organelle crosstalk provides critical insights into kidney disease pathogenesis and identifies novel targets for therapeutic intervention in AKI-to-CKD transition. Full article

Cysteine proteases (bromelain, ficin, and papain) are widely used in biotechnology and medicine, but their application is limited by rapid autolysis and oxidative inactivation. This study aimed to develop effective supports for these enzymes based on graft copolymers of sodium alginate and poly(N-vinylpyrrolidone) (SA-g-PVP) and to elucidate the structure–property relationships governing immobilization efficiency, catalytic activity, and storage stability. Copolymers were synthesized via radical solution polymerization under optimized conditions. Enzymes were immobilized by physical adsorption, and the resulting complexes were characterized by Fourier-transform infrared (FTIR) spectroscopy, protein content assays, proteolytic and amidase activity measurements, and molecular docking. The graft copolymer with a smaller particle size in solution provided a larger accessible surface area, leading to higher bromelain and papain loading. Ficin showed the opposite trend due to its unique surface amino acid composition. Immobilization dramatically increased storage stability: half-life values for bromelain, ficin, and papain reached up to 20, 14, and 14 days, respectively, compared to 1–3 days for the free enzymes. Molecular docking revealed that the dense polymer shell stabilizes the enzyme tertiary structure by forming multiple contacts with internal cavities and tunnels, thereby preventing autolysis and conformational unfolding. Collectively, these findings demonstrate that SA-g-PVP copolymers are promising, non-toxic supports for cysteine proteases, with ficin showing up to 100% activity recovery, making them suitable for food, cosmetic, and biomedical applications. Full article

Radiotherapy remains a central component of standard treatment for glioblastoma (GBM), yet recurrence is common because GBM radioresistance is reinforced by enhanced DNA damage repair, glioma stem cells (GSCs), hypoxia, extracellular matrix remodeling, and an immunosuppressive tumor microenvironment. FLASH radiotherapy (FLASH-RT), delivered at ultra-high dose rates, has shown reproducible normal-tissue-sparing effects in preclinical models, including the brain. In GBM models, however, available evidence indicates that FLASH-RT generally preserves tumor control at levels comparable to conventional radiotherapy rather than providing clearly superior eradication of hypoxic or stem-like tumor compartments. In parallel, endoplasmic reticulum (ER)-targeted interventions have emerged as a candidate strategy for disturbing tumor proteostasis, modulating unfolded protein response (UPR) signaling, impairing synthesis of repair-associated proteins, and promoting immunogenic cell death. This narrative review summarizes representative mechanisms of GBM radioresistance, appraises the opportunities and limitations of FLASH-RT in intracranial disease, and explains why ER targeting is discussed here as a lead but unproven biological axis for radiosensitization. We further compare ER-directed approaches with mitochondrial-, lysosomal-, and delivery-enabled radiosensitization strategies, and outline the translational variables that would determine clinical testability, including beam modality, blood–brain barrier heterogeneity, pharmacokinetics, treatment sequencing, and biomarker development. In this review, “physical precision” refers primarily to dose-rate-driven ultra-rapid delivery and the possibility of widening the normal-tissue therapeutic window under FLASH conditions, rather than to a universal depth–dose advantage shared by all FLASH platforms. Direct experimental evidence for combining FLASH-RT with ER-targeted therapy in GBM is currently lacking. We therefore present this model as a hypothesis-generating conceptual and translational framework for future preclinical testing rather than as an established therapeutic advance. Full article

Tyrosinase-related protein 1 (Tyrp1) is a melanosomal glycoprotein required for eumelanin biosynthesis through the oxidation of 5,6-dihydroxyindole-2-carboxylic acid (DHICA). Pathogenic variants in Tyrp1 cause oculocutaneous albinism type 3 (OCA3), but the molecular basis by which individual substitutions impair Tyrp1 stability and activity remains incompletely understood. Here, we examined wild-type Tyrp1 and three missense variants associated with OCA3: R356Q and R326H as OCA3-related variants, and D308N as a benign control; these were under conditions relevant to melanosome maturation. To assess stability, we developed a urea-induced unfolding assay in which His-tagged Tyrp1 variants were immobilized to Ni-NTA magnetic beads before chemical denaturation. R356Q was the most destabilized variant, with a ΔΔG of 0.695 kcal/mol at pH 5.0 (acidic conditions) and 1.998 kcal/mol at pH 7.4 (near-neutral conditions) relative to wild-type. R326H showed intermediate destabilization, whereas D308N behaved similarly to wild-type. DHICA oxidation assays in the presence of MBTH showed about 20% reduced catalytic activity for R356Q, particularly under acidic conditions. Molecular dynamics simulations and ligand docking were consistent with these findings and indicated that R356Q increases conformational flexibility and perturbs structural integrity. In contrast, glycosylation reduced conformational fluctuations and enhanced stability across Tyrp1 and mutant variants examined. Together, these results show that pH, glycosylation, and disease-associated substitutions collectively modulate Tyrp1 folding energetics and catalytic competence and identify R356Q as a strongly destabilizing OCA3 variant. By defining how disease-associated Tyrp1 substitutions affect protein stability and function, this study may provide a framework for interpreting genotype–phenotype relationships and improving molecular diagnosis of OCA3. Full article

Cationic antimicrobial peptides (AMPs) that can target multidrug-resistant pathogenic bacteria via multiple mechanisms are considered promising alternatives to antibiotics. Small (~9 kDa) highly acidic acyl carrier proteins (ACPs), which are a well-known cofactor protein in bacterial fatty acid synthesis (FAS), are a potential intracellular target for AMPs. A previous study has demonstrated that the human AMP LL-37 can bind to ACP and thereby affect FAS and the bacterial membrane integrity. In this work, we have investigated the interactions of different classes of AMPs and antibiofilm peptides (ABPs) with the ACPs of two pathogens. We first studied the folding characteristics of the two ACPs and found that Pseudomonas aeruginosa ACP (PaACP) is fully folded at neutral pH in the absence of divalent cations. On the other hand, the homologous Francisella novicida ACP (FnACP) is unfolded at low ionic strength, but it adopts a fully folded conformation after the addition of divalent cations such as Ca²⁺ or Mg²⁺. These distinct characteristics were shown to be related to a unique His residue that is involved in a stabilizing cation–π interaction. Subsequent biophysical SPR and NMR interaction studies reveal that cationic AMPs and ABPs such as LL-37, melittin, tritrpticin, indolicidin, puroindoline A, lactoferricin B and IDR-1018, but not F5W-magainin 2, can bind to both apo- and holo-ACPs. Binding of Arg-rich peptides is preferred over their Lys-rich analogs. Interestingly, all the peptides bind to holo-ACP with higher affinity than to apo-ACP, which lacks the functionally important phosphopantothenate group. NMR peak intensity perturbation data reveal that helix II of ACP, which is known to be directly involved in complex formation with bacterial FAS enzymes, acts as a common and main recognition site for the peptides. We propose that binding of AMPs and ABPs to this region of bacterial ACPs can directly block fatty acid synthesis and interfere in other ACP-dependent biosynthetic and regulatory events, which in turn could contribute to killing the bacteria and could also intervene in biofilm formation. Full article

Epidermal lipid homeostasis is crucial for skin barrier integrity. This study investigated the effects of an aqueous extract from Beta vulgaris subsp. vulgaris Beetroot Group (BvE) on stress responses and lipid metabolism in HaCaT keratinocytes. BvE, obtained from leaves grown in SETIS^® bioreactors as a standardized biomass source, was chemically characterized by ¹H NMR and ¹³C NMR. HaCaT cells were treated with BvE (1 µg/mL), H₂O₂, or palmitic/oleic acids (PA/OA) to evaluate its protective effects against oxidative damage and lipotoxic stress. Under these conditions, BvE exhibited a distinctive dual action as a reactive oxygen species (ROS) scavenger and triacylglycerol (TAG)-lowering agent. On the one hand, BvE was associated with decreased intracellular ROS levels and changes in NRF2 protein expression, suggesting involvement of redox-regulatory pathways. On the other hand, it was associated with attenuation of lipotoxicity, as evidenced by reduced lipid droplet (LD) formation and decreased expression of DGAT1 and PLIN2. Furthermore, these effects were accompanied by a reduction in Unfolded Protein Response (UPR) markers, modulation of AMPK-associated signaling, attenuation of mitochondrial disfunction, and decreased p53 phosphorylation, findings collectively consistent with a coordinated cytoprotective response. In conclusion, BvE shows potential to protect keratinocytes against lipotoxicity and oxidative stress through mechanisms that may involve both chemical and biological antioxidant activity and metabolic reprogramming, supporting its further investigation for dermatological applications. Full article

Background: T-cell acute lymphoblastic leukemia (T-ALL) remains a challenging malignancy with limited targeted therapies. Natural phenanthrene derivatives represent a promising source of antileukemic agents. Objective: We screened a library of natural phenanthrene-type compounds to identify cytotoxic leads in Jurkat T-ALL cells and investigated the mechanisms underlying their activity, including potential synergy with the proteasome inhibitor bortezomib (BTZ). Methods: Jurkat cells were treated with thirteen natural compounds at 10 and 20 µM for 48 h; cell viability was assessed by WST-1 cell viability assay. Dose–response curves were generated to calculate IC₅₀ values. Apoptosis was evaluated by Hoechst 33342/PI staining and Annexin V/PI flow cytometry. Synergy with BTZ was analyzed using a fixed-ratio combination index (CI) approach and IC₅₀ shift analysis. ER stress signaling was characterized by Western blotting, quantitative RT-PCR of UPR genes (GRP78, ATF6), and immunoprecipitation of GRP78 followed by ubiquitin immunoblotting. Results: Among the compounds screened, Cypripedin showed the most potent cytotoxicity with an IC₅₀ of 6.52 µM. It induced a dose-dependent increase in apoptosis. Combination with BTZ yielded a CI < 0.5 and reduced BTZ IC₅₀ from 3.43 to 1.88 ng/mL. Cypripedin activated the unfolded protein response (UPR), modulated key ER stress markers including GRP78, p-PERK, p-eIF2α, p-JNK, and ATF6, downregulated UPR gene transcripts, and promoted GRP78 ubiquitination. Molecular docking predicted strong binding of Cypripedin to the GRP78 ATPase domain (Vina score −7.630 kcal/mol), supporting its mechanism of action. Conclusion: Cypripedin induces apoptosis in Jurkat T-ALL cells, synergizes with BTZ, and modulates ER stress through GRP78 ubiquitination. These findings support its further development as a potential T-ALL therapeutic. Full article

Gestational diabetes mellitus (GDM) is increasingly recognized as a complex pathology rooted in systemic and organelle-level dysfunction, specifically involving chronic low-grade inflammation (CLGI), mitochondrial impairment, and endoplasmic reticulum (ER) stress. Central to this pathophysiology is mitochondrial dysfunction, characterized by reduced respiration, impaired metabolic flexibility, and dysregulated fission/fusion machinery, which fuels a self-perpetuating cycle of reactive oxygen species (ROS) production. Concurrently, chronic ER stress triggered by hyperglycemia and lipotoxicity activates the unfolded protein response (UPR), further amplifying redox imbalance through the Endoplasmic Reticulum Oxidoreductin 1/Protein Disulfide Isomerase (ERO1/PDI) axis and bridging metabolic toxicity to inflammation via c-Jun N-terminal kinase (JNK) and nuclear factor kappa-light-chain–enhancer of activated B cells (NF-κB) signaling. The Advanced Glycation Endproducts (AGEs) and the Receptor for Advanced Glycation Endproducts (RAGE) axis act as a molecular catalyst that sequester antioxidants and drive pro-inflammatory feedback loops. These converging mechanisms culminate in profound placental maladaptation, including structural abnormalities like chorangiosis and functional defects in nutrient transport mediated by hyperactive mechanistic target of rapamycin complex 1 (mTORC1) signaling. This review article provides insight into recent evidence to elucidate the meta-inflammatory environment of GDM, where modest but sustained elevations in biomarkers like Interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α) disrupt redox homeostasis and impair insulin signaling pathways through the activation of stress-sensitive kinases. By integrating these molecular perspectives, the article underscores the necessity of targeting the systemic inflammatory and oxidative continuum spanning pre-conception to the antenatal period through lifestyle interventions and emerging therapeutic strategies to mitigate GDM risk and improve maternal–fetal outcomes. Full article

To maintain homeostatic conditions and optimal function during stressors, mitochondria initiate retrograde signaling. The mitochondrial integrated stress response (ISR) and unfolded protein response (UPR^mt) are critical quality control mechanisms activated during instances of mitochondrial perturbations. Restoration of mitochondrial homeostasis is orchestrated by three transcription factors, ATF4, CHOP, and ATF5, which upregulate protective genes to counteract stress. As the health and function of skeletal muscle are heavily dependent on a highly adaptive mitochondrial network, defining how mitochondrial health is maintained across various conditions is essential. Although several studies demonstrate the importance of these responses following instances of stress, the signaling mechanisms required to initiate such pathways remain poorly characterized in skeletal muscle. This review examines how the mitochondrial ISR/UPR^mt and related transcription factors respond to organellar stress by emphasizing the molecular events that occur during exercise, aging and muscle disuse. By consolidating the literature, this work aims to highlight the current understanding of mitochondrial stress response signaling within skeletal muscle and thus emphasize areas for future research and potential therapeutic strategies during divergent metabolic conditions. Full article

Prostate cancer (PCa) remains lethal at advanced stages, partly due to stem-like subpopulations known as prostate cancer stem cells (PCSCs) that sustain tumor growth and therapeutic resistance. Imipridones are small-molecule anticancer agents, with next-generation derivatives ONC206 and ONC212 designed for enhanced potency and broader activity. This study compared their antitumor efficacy and mechanisms in advanced androgen-independent PCa (AIPC) models, namely DU145 and PC3 cells, using two- and three-dimensional systems encompassing bulk cancer cells and PCSCs. DU145 and PC3 AIPC cells were treated with ONC201 (parent compound), ONC206, or ONC212. Functional assays assessed proliferation, viability, migration, invasion, PCa spheroids formation, cell cycle distribution, and mitochondrial membrane potential and mass, while RNA sequencing defined transcriptional responses. ONC212 was the most potent derivative, inhibiting proliferation and migration and abolishing PCa spheroids at nanomolar doses, whereas ONC201 and ONC206 required higher concentrations. Transcriptomic analyses revealed shared repression of DNA replication and cell-cycle transition programs, with activation of integrated stress and unfolded protein responses (ISR/UPR) and FOXO signaling. ONC206 favored PERK–ATF4-mediated apoptosis with reduced DNA repair, while ONC212 more strongly impacted oxidative phosphorylation-related pathways and mitochondrial RNA processing. Imipridones induced a time-dependent cell-cycle redistribution with increased sub-G1 accumulation and modulated mitochondrial membrane potential and mass in a context-dependent manner. Collectively, these findings position ONC212 as a leading imipridone candidate in AIPC models, combining potent inhibition of tumor and stem-like cell functions with a coherent stress-response signature that supports further translational evaluation. Full article

The present study aimed to compare the composition structure, interfacial, and antioxidant activities of flaxseed protein isolates (FPIs) in different flaxseed varieties. The results showed that apparently intact protein bodies (PBs) were manifested as densely staining cytoplasmic inclusions with distinct boundaries and varying diameter ranges among different flaxseed varieties. Through alkali extraction with isoelectric precipitation, FPIs exhibited a relatively small and irregular lamellar strip structure with varying sizes and shapes packed with spherical particles in studied flaxseed varieties. The different composition structures of FPIs among studied flaxseed varieties were also obtained, involving the protein subunits’ intrinsic fluorescence properties, secondary structures, and amino acid profiles. These structural differences also led to differential purities, aqueous solubility, dispersion properties, and surface charges. Moreover, the varying emulsifying and foaming properties of FPIs from different flaxseed varieties were also observed due to the formation of coarse lipid droplets (5~40 μm) and foams (20~100 μm) with the specific structure of the oil/gas–water interface and bulk aqueous phase. The retention of phenolic compounds into FPIs still displayed evident variety specificity from 323 to 478 mg/100 g and 210 to 347 mg/100 g, which definitely led to escalated antioxidant activities. Thus, FPIs from Longya 13# and Neiya 9# flaxseed varieties were screened for favorable emulsifying and foaming properties due to the balanced molecular rigidity/unfolding and interfacial adsorption/stabilization behavior. Full article