Search Results (167)

Extracellular signal-regulated kinase 1/2 (ERK1/2) inhibitors show therapeutic potential in triple-negative breast cancer (TNBC), but resistance through compensatory signaling limits their efficacy. We previously identified the secretory carrier membrane protein 3 (SCAMP3) as a regulator of TNBC progression and ERK1/2 activation. Here, we investigated the role of SCAMP3 in ERK1/2 signaling and therapeutic response using TMT-based LC-MS/MS phosphoproteomics of wild-type (WT) and SCAMP3 knockout (SC3KO) SUM-149 cells under basal conditions, after epidermal growth factor (EGF) stimulation, and during ERK1/2 inhibition with MK-8353. A total of 4408 phosphosites were quantified, with 1093 significantly changed. SC3KO abolished residual ERK activity under MK-8353 and affected the compensatory activation of oncogenic pathways observed in WT cells. SC3KO reduced the phosphorylation of ERK feedback regulators RAF proto-oncogene serine/threonine-protein kinase Raf-1 (S43) and the dual-specificity mitogen-activated protein kinase kinase 2 (MEK2) (T394), affected other ERK targets, including nucleoporins, transcription factors, and metabolic enzymes triosephosphate isomerase (TPI1) (S21) and ATP-citrate lyase (ACLY) (S455). SCAMP3 loss also impaired the mammalian target of rapamycin complex I (mTORC1) signaling and disrupted autophagic flux, evidenced by elevated sequestosome-1 (SQSTM1/p62) and microtubule-associated protein light chain 3 (LC3B-II) with reduced levels of the autophagosome lysosome maturation marker, Rab7A. Beyond ERK substrates, SC3KO affected phosphorylation events mediated by other kinases. These findings position SCAMP3 as a central coordinator of ERK signaling and autophagy. Our results support SCAMP3 as a potential therapeutic target to enhance ERK1/2 inhibitor clinical efficacy and overcome adaptive resistance mechanisms in TNBC. Full article

Triple-negative breast cancer (TNBC) is an aggressive, heterogeneous subtype of breast cancer. miRNAs play an essential role in TNBC pathogenesis and prognosis. Obesity is linked with an increased risk for several cancers, including breast cancer. Obesity is also related to the dysregulation of miRNA expression in adipose tissues. However, there is limited knowledge about race- and obesity-specific differential miRNA expression in TNBC. We performed miRNA sequencing of 48 samples (24 tumor and 24 adjacent non-tumor tissues) and RNA sequencing of 24 tumors samples from Black (AA) and White (EA) TNBC patients with or without obesity. We identified 55 miRNAs exclusively associated with tumors in obese EA patients and 33 miRNAs in obese AA patients, each capable of distinguishing tumor tissues from obese from lean individuals within their respective racial groups. In EA, we detected 41 significant miRNA–mRNA correlations. Notably, miR-181b-5p and miR-877-5p acted as negative regulators of tumor-suppressor genes (e.g., HEY2, MCL2, HAND2), while miR-204-5p and miR-143-3p appeared to indirectly target oncogenes (e.g., RAB10, DR1, PTBP3, NCBP1). Among AA patients, we found 28 significant miRNA–mRNA interactions. miR-195-5p, miR-130a-3p, miR-130a-5p, miR-424-5p, miR-148a-3p, miR-374-5p, and miR-30a-5p each potentially downregulated two or more genes (e.g., CLCN4, PLCB1, CDC25B, AEBP2, ERBB4). Pathway enrichment analysis highlighted KRAS, ESR1, ESR2, RAB10, TNRC6C, and NCAN as the most commonly differentially expressed in EA, whereas ERBB4, PLCB1, and SERPINE1 were most frequently in AA. These findings highlight the importance of considering race-specific miRNA–mRNA signatures in understanding TNBC in the context of obesity, offering insights into biomarker-driven patient stratification for targeted therapeutic strategies. Full article

Salivary gland dysfunction is a common but underexplored complication of menopause that contributes to oral dryness, dysphagia, and increased risk of infection. Although ferroptosis, a form of regulated necrotic cell death driven by iron-dependent lipid peroxidation, has recently been implicated in postmenopausal tissue degeneration, its regulatory mechanisms in salivary glands remain unclear. In this study, we investigated the roles of mitochondrial dysfunction and mitophagy in driving ferroptosis-induced salivary gland injury in an ovariectomized (OVX) rat model of estrogen deficiency. OVX rats exhibited elevated markers of oxidative stress, lipid accumulation, and iron overload, and suppression of GPX4 activity in the salivary glands, consistent with ferroptotic activation. These changes were accompanied by impaired mitochondrial dynamics (MFN1 and OPA1), decreased expression of mitochondrial antioxidant regulators (PGC-1α, SOD, and catalase), and upregulation of mitophagy-related genes (PINK1, ULK1, Rab9, and LC3B), as well as LAMP, a lysosomal marker involved in autophagosome–lysosome fusion, while ferritinophagy (NCOA4) remained unchanged. Early administration of ferrostatin-1 effectively suppressed these pathological changes, preserving both glandular structure and function, as evidenced by the restored AQP5 and AMY2A expression. Collectively, our findings reveal that ferroptosis in estrogen-deficient salivary glands is regulated by mitochondrial instability and aberrant mitophagy, and ferrostatin-1 mitigates this cascade through multi-level mitochondrial protection. These results highlight ferrostatin-1 as a promising preventive agent against menopause-associated salivary gland dysfunction, with broader implications for organ-specific ferroptosis modulation. Full article

RNA-seq analysis of the highly differentiated human retinal pigment epithelial (RPE) cell-line ARPE-19, cultured on transwells for ≥4 months, yielded 44,909 genes showing 83.35% alignment with the human reference genome. These included mRNA transcripts of RPE-specific genes and those involved in retinopathies. Monolayers were fed photoreceptor outer segments (POS), designed to be synchronously internalised, mimicking homeostatic RPE activity. Cells were subsequently fixed at 4, 6, 24 and 48 h when POS were previously shown to maximally co-localise with Rab5, Rab7, LAMP/lysosomes and LC3b/autophagic compartments. A comprehensive analysis of differentially expressed genes involved in proteolysis revealed a pattern of gene orchestration consistent with POS breakdown in the autophagy-lysosomal pathway. At 4 h, these included elevated upstream signalling events promoting early stages of cargo transport and endosome maturation compared to RPE without POS exposure. This transcriptional landscape altered from 6 h, transitioning to promoting cargo degradation in autolysosomes by 24–48 h. Longitudinal scrutiny of mRNA transcripts revealed nuanced differences even within linked gene networks. POS exposure also initiated transcriptional upregulation in ubiquitin proteasome and chaperone-mediated systems within 4–6 h, providing evidence of cross-talk with other proteolytic processes. These findings show detailed evidence of transcriptome-level responses to cargo trafficking and processing in RPE cells. Full article

The phosphorylation of Rab10 (pT73-Rab10) by LRRK2 promotes the establishment of epithelial cell polarity by controlling the trafficking to the primary cilia membrane of cilia-resident proteins and signaling proteins. Previous studies have identified a site in the globular tail domain of MYO5A that specifically binds to only the phosphorylated form of Rab10. In this work, we have demonstrated that pT73-Rab10 does not associate with the globular tail of MYO5B. We have mapped the putative binding site to a required three amino acids (MEN, 1473–1475) in the MYO5A globular tail domain that are not found in the MYO5B globular tail. Substitution of the MEN amino acid sequence found in MYO5A into the paralogous position in the MYO5B globular tail conferred the ability to associate with pT73-Rab10. The results demonstrate that the interactors with MYO5A and MYO5B are not completely overlapping and that the interaction of pT73-Rab10 is specific to the MYO5A globular tail domain. Full article

Background: Previous proteomic studies provided valuable information about cataracts, but unclarified issues, such as sex and ethnicity-associated differences, remain. This study aimed to provide additional data on cataract-related proteins regarding age, sex, and cataract type. Methods: Twenty-six female and seven male Turkish cataract patients were screened for visual acuity and dysfunctional lens index. A nano-LC-MS/MS system and Progenesis QI software v3.0 were used for protein identification and quantification. The remaining data were evaluated with SPSS Version 29.0 software. Results: Proteins that showed age-associated changes were mainly involved in cytoskeletal organization. A glyoxalase enzyme, caveolin 1, and HS90B were lower, and RAB8B and ATP6V1B1 were higher in lenses in women. Proteins with lower levels in cataractous lenses than in transparent lenses included filensin and phakinin, concurrent with previous publications, and LCTL, GDI, HSPB1, and EIF4A2, not reported before. Corticonuclear cataracts constituted the only group showing depletions in putatively protective proteins, while the cortical type was the least influenced. ANXA1 and DNHD1 positively, and TCPD, SEC14L2, and PRPS1 proteins negatively correlated with visual acuity. Conclusions: This study revealed cataract-related proteins concurrent with earlier studies and new ones hitherto unreported. Despite the low number of patients investigated, the results merit further research, as these new proteins are highly likely to be involved in cataractogenesis. Full article

Advancing age in men significantly contributes to declining sperm fertility. Information on age-related proteomic changes in spermatozoa is limited. This study involved normal fertile Arab men in three age groups: young adult (21–30 years; n = 6), late adult (31–40 years; n = 7), and advanced age (40–51 years; n = 5). Gradient-purified spermatozoa were analyzed using LC-MS/MS and proteomic data were processed using Progenesis QI (QIfp) v3.0 and UniProt/SwissProt. Significantly enriched annotations and clustering of proteins in the proteomic datasets were identified (2-fold change; p < 0.05). A total of 588 proteins were identified, with 93% shared across the three groups. Unique proteins were MYLK4 for the young adult group, PRSS57 for the late adult group, and HMGB4, KRT4, LPGAT1, OXCT2, and MGRN1 for the advanced age group. Furthermore, 261 (44%) proteins were differentially expressed (p < 0.05) across the three groups. Functional enrichment analysis suggested an aging-related significant increase in pathways associated with neurodegenerative diseases and protein folding, alongside decreases in glycolysis/gluconeogenesis, flagellated sperm motility, acetylation, phosphoprotein modifications, oxidation processes, and Ubl conjugation. Cluster analysis highlighted significantly upregulated proteins in young adults (e.g., H2BC1, LAP3, SQLE, LTF, PDIA4, DYNLT2) and late adults (e.g., ATP5F1B, ODF2, TUBA3C, ENO1, SPO11, TEX45, TEKT3), whereas most proteins in the advanced age group exhibited downregulation (e.g., SPESP1, RAB10, SEPTIN4, RAB15, PTPN7, USP5, ANXA1, PRDX1). In conclusion, this study revealed aging-associated proteomic changes in spermatozoa that impact critical processes, including spermatogenesis, motility, metabolism, and fertilization, potentially contributing to fertility decline. These changes provide a molecular framework for developing therapies to preserve sperm proteostasis and enhance fertility in older men. Full article

The unique secondary messenger 2′3′-cGAMP, produced by cGAS in response to cytosolic dsDNA, plays a critical role in activating innate immunity by binding to and activating STING via cell-intrinsic, autocrine, or paracrine mechanisms. Recently, we identified Rab18 as a novel, STING-independent binder of 2′3′-cGAMP. Binding of 2′3′-cGAMP to Rab18 promotes Rab18 activation and induces cell migration. However, the downstream mechanisms by which 2′3′-cGAMP-induced Rab18 activation regulates cell migration remain largely unclear. Herein, using phospho-profiling analysis, we identify MAPK signaling as a key downstream effector of the 2′3′-cGAMP/Rab18 axis that promotes the expression of FosB2 and drives cell migration. Furthermore, we identify MMP3 as a major transcriptional target of FosB2, through which the 2′3′-cGAMP/Rab18/MAPK/FosB2 signaling pathway positively regulates cell migration. Together, our findings provide new mechanistic insights into how 2′3′-cGAMP signaling controls cell migration and suggest the potential of MAPK inhibitors to block 2′3′-cGAMP-induced migratory responses. Full article

Disorders of vesicular trafficking and genetic defects in autophagy play a critical role in the development of metabolic and neurometabolic diseases. These processes govern intracellular transport and lysosomal degradation, thereby maintaining cellular homeostasis. In this article, we present two siblings with a novel homozygous variant in VPS51 (Vacuolar protein sorting 51) gene (c.1511C>T; p.Thr504Met), exhibiting developmental delay, a thin corpus callosum, severe intellectual disability, epilepsy, microcephaly, hearing loss, and dysphagia. This study aimed to investigate the effects of the novel VPS51 gene variation at the RNA and protein level in fibroblasts derived from patients. A comparative proteomic analysis, which has not been previously elucidated, was performed to identify uncharacterized proteins associated with vesicular trafficking. Furthermore, the impact of disrupted pathways on mitochondria–lysosome contact sites was assessed, offering a thorough pathophysiological evaluation of GARP/EARP (Golgi Associated Retrograde Protein / Endosome Associated Retrograde Protein) complex dysfunction. An analysis of mRNA expression indicated decreased levels of the VPS51 gene, alongside modifications in the expression of autophagy-related genes (LC3B, p62, RAB7A, TBC1D15). Western blotting demonstrated a reduction in VPS51 and autophagy-related protein levels. Proteomic profiling revealed 585 differentially expressed proteins, indicating disruptions in vesicular trafficking, lysosomal function, and mitochondrial metabolism. Proteins involved in mitochondrial β-oxidation and oxidative phosphorylation exhibited downregulation, whereas pathways related to glycolysis and lipid synthesis showed upregulation. Live-cell confocal microscopy revealed a notable increase in mitochondria–lysosome contact sites in patient fibroblasts, suggesting that VPS51 protein dysfunction contributes to impaired organelle communication. The findings indicate that the novel VPS51 gene variation influences intracellular transport, autophagy, and metabolic pathways, offering new insights into its involvement in neurometabolic disorders. Full article

Multiple sclerosis (MS) is a chronic inflammatory, neurodegenerative disease with yet-unresolved mechanisms of progression. To address MS severity and neurological deficits, we analyzed seven potentially functional genetic variants and their haplotypes in 845 MS patients. Based on our previous results of targeted RNAseq on ferroptosis-related genes in distinctive MS phenotypes, we selected putative regulatory variants in the top three DEGs (CDKN1A, MAP1B and EGLN2) and investigated their association with gene expression, plasma/serum parameters and disease severity (EDSS, MSSS, gARMSS). The study included 604 patients with relapsing–remitting (RR) and 241 with progressive (P) MS. The variants CDKN1A rs3176326 and rs3176336, EGLN2 rs111833532, MAP1B rs62363242 and rs1217817 with the previously reported DYSF-ZNF638 locus rs10191329, and MTSS1 rs9643199 were genotyped using TaqMan^®, and the HLA-DRB1*15:01 status was also determined. Significant association of the rare MAP1B rs62363242 allele with PMS in females, independent of HLA-DRB1*1501, was found. The A allele-containing genotypes were associated with molecular components of iron metabolism. CDKN1A haplotypes were significantly associated with CDKN1A mRNA levels in RRMS and SPMS patients. RAB4B-EGLN2 locus rs111833532 and DYSF-ZNF638 locus rs10191329 showed significant associations with EDSS, MSSS and gARMSS. We detected haplotypes associated with the expression of CDKN1A, a part of the p53-p21 axis known to affect T cell activation/proliferation. RAB4B-EGLN2, an oxygen sensor and critical regulator of the response to hypoxia, variant rs111833532, along with DYSF-ZNF638 locus rs10191329, was associated with clinical severity. The indicated, novel, sex-specific association of MAP1B rs62363242 with the course of MS remains to be validated in larger studies. Full article

The selection of appropriate reference genes is critical for standardizing quantitative real-time reverse transcription polymerase chain reaction (RT-qPCR) data, thereby ensuring accurate and reliable results of gene expression analysis. In this study, we identified 10 candidate reference genes (encoding α-tubulin, G6PDH, Rab1, RT, RPS13, β-tubulin, DPH1, HSP90, GAPDH, and CP) and evaluated their suitability for use as reference genes in the pest insect, Bactrocera tau. Analysis was conducted using three software-based methods—Delta CT, NormFinder, and BestKeeper—alongside the online tool RefFinder. Expression levels of these genes were analyzed across various B. tau developmental stages and body parts. The overall ranking of reference gene stability scores was as follows: α-tubulin > G6PDH > CP > β-tubulin > RT > HSP90 > GAPDH > DPH1 > RPS13 > Rab1. Ultimately, α-tubulin and G6PDH were identified as the most stable reference genes for B. tau. Full article

Background: Macrocephaly can be a component manifestation of several monogenic conditions, in association with intellectual disability/developmental delay (ID/DD) behaviour abnormalities, including autism spectrum disorders (ASD), and variable additional features. On the other hand, idiopathic ASD can present with developmental delay and macrocephaly. Methods: We carried out a retrospective analysis of a cohort of 78 patients who were tested from February 2017 to December 2024 by high-throughput sequencing of a panel of 27 genes (ABCC9, AKT1, AKT2, AKT3, BRWD3, DIS3L2, DNMT3A, EZH2, GPC3, GPC4, HERC1, MED12, MTOR, NFIA, NFIX, NSD1, PDGFRB, PIK3CA, PIK3R1, PIK3R2, PPP2R1A, PPP2R5D, PTEN, RAB39B, RNF135, SETD2, and TBC1D7) because of neurodevelopmental impairment, including ID/DD, ASD/behaviour abnormalities associated with macrocephaly, mimicking to a large extent idiopathic ASD. Results: Pathogenic variants leading to the diagnosis of monogenic conditions were detected in 22 patients (28%), including NSD1 (2), PTEN (16), and PPP2R5D (4). Distinctive of the PTEN-associated phenotype were true macrocephaly (100%), ASD or behaviour abnormalities (92%), mild/borderline ID (79%), and no facial dysmorphisms. Typical of the PPP2R5D-associated phenotype were relative macrocephaly (75%), a few unspecific peculiar facial characteristics (50%), and a more variable presentation of the neurodevelopmental phenotype. Conclusions: Pathogenic variants in PTEN and PPP2R5D are the most recurrent gene mutations in a patient-oriented procedure for the genetic diagnosis of apparently idiopathic ASD and behaviour abnormalities associated with macrocephaly. The clinical applicability of the presented diagnostic strategy is discussed. Full article

Regenerative processes occur at various levels in all organisms, yet their complexity continues to raise new questions about their mechanisms. It has been demonstrated that small extracellular vesicles (sEVs), secreted by all cells and influencing their function, play a significant role in regeneration. In the context of regenerative processes, oral mucosal tissues consistently receive interest, as they are among the most rapidly healing tissues in the human body. In this study, we utilized spatial transcriptomics to map gene expression to specific spatial locations within the gingiva tissue section, using publicly available transcriptomic data. This analysis revealed new insights into this tissue and the biogenesis of sEVs within it. The identified clusters encompassed two main regions—the epithelium and lamina propria—as well as minor niches within them. Using Gene Ontology (GO) analysis, we identified two clusters most enriched in extracellular vesicle-related GO processes. These included the superficial and deeper layers of the sulcular epithelium, one of the most peripheral regions of the gingiva. Of the 43 genes identified in the literature as having a potential or documented role in sEVs biogenesis, 12 were selected for further analysis. MUC1, SDCBP2, and VPS37B showed clear specificity and the highest expression in the superficial layer of the sulcular epithelium. CHMP4C also exhibited high expression in this layer, though its levels were comparable to the outer layer of the oral epithelium. Other well-established sEVs marker genes, such as ANXA2, CD9, CD63, CD81, FLOT1, RAB22A, RAB27B, and RAB5A, were also expressed in the examined tissue; however, their expression was not specifically exclusive to the sulcular epithelium. Our study is the first to perform a meta-analysis of available gingival transcriptomic data in the specific context of sEVs biogenesis. The presented data and conclusions provide new insights into the role of different structures within healthy human gingiva and shed new light on both known and potential markers of sEVs biogenesis. These findings may contribute to the development of regeneration-targeted research, especially on oral tissues. Full article

Objective: Lop sheep species exhibit remarkable adaptability to desert pastures and extreme arid climates, demonstrating tolerance to rough feeding and high resistance to stress. However, little is known about the population genetic diversity of Lop sheep and the genetic mechanisms underlying their adaptability to extreme environments. Methods: Blood samples were collected from a total of 110 individuals comprising 80 Ruoqiang Lop sheep and 30 Yuli Lop sheep. A total of 110 Lop sheep were subjected to whole genome resequencing to analyze genetic diversity, population structure, and signatures of selection in both regions. Results: The genetic diversity of the Lop sheep population is substantial, and the degree of inbreeding is low. In comparison to the Lop sheep in Yuli County, the genetic diversity and linkage disequilibrium analysis results for the Lop sheep population in Ruoqiang County are slightly lower. Population structure analysis indicates that Ruoqiang and Yuli Lop sheep have differentiated into two independent groups. Using Yuli Lop sheep as the reference group, an analysis of the extreme environmental adaptability selection signal of Lop sheep was conducted. The FST and π ratio under the 1% threshold identified 1686 and 863 candidate genes, respectively, with their intersection yielding a total of 122 candidate genes. Functional annotation revealed that these genes are associated with various traits, including immune response (SLC12A2, FOXP1, PANX1, DYNLRB2, RAP1B, and SEMA4D), heat and cold resistance (DNAJC13, PLCB1, HIKESHI, and PITPNC1), desert adaptation (F13A1, PANX1, ST6GAL1, STXBP3, ACTN4, and ATP6V1A), and reproductive performance (RAP1B, RAB6A, PLCB1, and METTL15). Conclusions: These research findings provide a theoretical foundation for understanding the survival and reproductive characteristics of Lop sheep in extreme environments, and they hold practical value for the conservation and utilization of Lop sheep genetic resources, as well as for genetic improvement efforts. Full article

Autophagy is a critical mechanism by which methamphetamine (METH) induces neuronal damage and neurotoxicity. Prolonged METH exposure can result in the accumulation of autophagosomes within cells. The autophagy process encompasses several essential vesicle-related biological steps, collectively referred to as the autophagic flux. However, the precise mechanisms by which METH modulates the autophagic flux and the underlying pathways remain to be elucidated. In this study, we utilized a chronic METH exposure mouse model and cell model to demonstrate that METH treatment leads to an increase in p62 and LC3B-II and the accumulation of autophagosomes in striatal neurons and SH-SY5Y cells. To assess autophagic flux, this study utilized autophagy inhibitors and inducers. The results demonstrated that the lysosomal inhibitor chloroquine exacerbated autophagosome accumulation; however, blocking autophagosome formation with 3-methyladenine did not prevent METH-induced autophagosome accumulation. Compared to the autophagy activator rapamycin, METH significantly reduced autophagosome–lysosome fusion, leading to autophagosome accumulation. Rab7a is a critical regulator of autophagosome–lysosome fusion. Although Rab7a expression was upregulated in SH-SY5Y cells and brain tissues after METH treatment, immunoprecipitation experiments revealed weakened interactions between Rab7a and the lysosomal protein RILP. Overexpression of active Rab7a (Rab7a Q67L) significantly alleviated the METH-induced upregulation of LC3-II and p62. PTEN, a key regulator of Rab7a dephosphorylation, was downregulated following METH treatment, resulting in decreased Rab7a dephosphorylation and reduced Rab7a activity, thereby contributing to autophagosome accumulation. We further investigated the role of neuronal exosomes in the autophagy process. Our results demonstrated that the miRNA expression profiles in exosomes released by METH-induced SH-SY5Y cells were significantly altered, with 122 miRNAs upregulated and 151 miRNAs downregulated. KEGG and GO enrichment analyses of these differentially expressed miRNAs and their target genes revealed significant associations with the autophagy pathway and potential regulation of PTEN expression. Our experiments confirmed that METH-induced exosomes reduced PTEN expression levels and decreased Rab7a dephosphorylation, thereby exacerbating autophagic flux impairment and autophagosome accumulation. In conclusion, our study indicated that METH and its induced neuronal exosomes downregulate PTEN expression, leading to reduced Rab7a dephosphorylation. This, in turn, hinders the fusion of autophagosomes and lysosomes, ultimately resulting in autophagic flux impairment and neuronal damage. Full article