A second-generation series of biscyclometalated 2-(5-aryl-thienyl)-benzimidazole and -benzothiazole Ir(III) dppz complexes [Ir(C^N)2(dppz)]+, Ir1-Ir4, were rationally designed and synthesized, where the aryl group attached to the thienyl ring was p-CF3C6H4 or p-Me2NC6H4. These new Ir(III) complexes were assessed as photosensitizers to explore the structure-activity correlations for their potential use in biocompatible anticancer photodynamic therapy. When irradiated with blue light, the complexes exhibited high selective potency across several cancer cell lines predisposed to photodynamic therapy; the benzothiazole derivatives (Ir1 and Ir2) were the best performers, Ir2 being also activatable with green or red light. Notably, when irradiated, the complexes induced leakage of lysosomal content into the cytoplasm of HeLa cancer cells and induced oncosis-like cell death. The capability of the new Ir complexes to photoinduce cell death in 3D HeLa spheroids has also been demonstrated. The investigated Ir complexes can also catalytically photo-oxidate NADH and photogenerate 1O2 and/or •OH in cell-free media.
- MeSH
- antitumorózní látky * farmakologie MeSH
- benzothiazoly MeSH
- fotosenzibilizující látky farmakologie terapeutické užití MeSH
- fototoxická dermatitida * farmakoterapie MeSH
- iridium farmakologie MeSH
- komplexní sloučeniny * farmakologie MeSH
- lidé MeSH
- lyzozomy MeSH
- nádorové buněčné linie MeSH
- nádory * farmakoterapie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
INTRODUCTION: The aging process is intricately linked to alterations in cellular and tissue structures, with the respiratory system being particularly susceptible to age-related changes. Therefore, this study aimed to profile the activity of proteases using activity-based probes in lung tissues of old and young rats, focusing on the expression levels of different, in particular cathepsins G and X and matrix Metalloproteinases (MMPs). Additionally, the impact on extracellular matrix (ECM) components, particularly fibronectin, in relation to age-related histological and ultrastructural changes in lung tissues was investigated. MATERIALS AND METHODS: Lung tissues from old and young rats were subjected to activity-based probe profiling to assess the activity of different proteases. Expression levels of cathepsins G and X were quantified, and zymography was performed to evaluate matrix metalloproteinases activity. Furthermore, ECM components, specifically fibronectin, were examined for signs of degradation in the old lung tissues compared to the young ones. Moreover, histological, immunohistochemical and ultrastructural assessments of old and young lung tissue were also conducted. RESULTS: Our results showed that the expression levels of cathepsins G and X were notably higher in old rat lung tissues in contrast to those in young rat lung tissues. Zymography analysis revealed elevated MMP activity in the old lung tissues compared to the young ones. Particularly, significant degradation of fibronectin, an essential ECM component, was observed in the old lung tissues. Numerous histological and ultrastructural alterations were observed in old lung tissues compared to young lung tissues. DISCUSSION AND CONCLUSION: The findings indicate an age-related upregulation of cathepsins G and X along with heightened MMP activity in old rat lung tissues, potentially contributing to the degradation of fibronectin within the ECM. These alterations highlight potential mechanisms underlying age-associated changes in lung tissue integrity and provide insights into protease-mediated ECM remodeling in the context of aging lungs.
- MeSH
- extracelulární matrix metabolismus ultrastruktura MeSH
- fibronektiny * metabolismus MeSH
- kathepsin G metabolismus MeSH
- krysa rodu rattus MeSH
- lyzozomy ultrastruktura metabolismus MeSH
- matrixové metaloproteinasy metabolismus MeSH
- plíce * ultrastruktura metabolismus MeSH
- proteasy metabolismus MeSH
- stárnutí * metabolismus MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- mužské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Rasopathies are genetic disorders often associated with developmental delay and intellectual disability. Noonan syndrome (NS) is one of the most common Rasopathies, caused by mutations in PTPN11 in more than 50% of cases. In mammalian neurons, PTPN11 controls the trafficking of postsynaptic glutamate receptors. This process is disrupted in neurons expressing PTPN11 variants associated with Rasopathies and is thought to contribute to the cognitive impairments in Noonan syndrome. Recent work revealed presynaptic impairments upon expression of RASopathy-linked PTPN11 variants in Drosophila. However, the presynaptic role of PTPN11 has not yet been addressed in mammals. Here, we investigated membrane trafficking of synaptic vesicles in cultured mouse cortical neurons expressing Rasopathy-associated PTPN11D61Y variant. We observed a significantly smaller readily releasable and total recycling pool of synaptic vesicles. The drop in synaptic vesicle release competence was accompanied by a decreased rate of SV retrieval. Interestingly, the presynaptic phenotype was evident in mature (DIV21) but not in immature (DIV12) neurons. Thus, our data reveal importance of balanced PTPN11 activity for normal trafficking of neurotransmitter-filled synaptic vesicles in the presynaptic ending of mature neurons.
- MeSH
- kultivované buňky MeSH
- mutace genetika MeSH
- myši MeSH
- neurony metabolismus MeSH
- stárnutí genetika metabolismus MeSH
- synaptické vezikuly * metabolismus MeSH
- tyrosinfosfatasa nereceptorového typu 11 * metabolismus genetika MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
The major organelles of the endomembrane system were in place by the time of the last eukaryotic common ancestor (LECA) (~1.5 billion years ago). Their acquisitions were defining milestones during eukaryogenesis. Comparative cell biology and evolutionary analyses show multiple instances of homology in the protein machinery controlling distinct interorganelle trafficking routes. Resolving these homologous relationships allows us to explore processes underlying the emergence of additional, distinct cellular compartments, infer ancestral states predating LECA, and explore the process of eukaryogenesis itself. Here, we undertake a molecular evolutionary analysis (including providing a transcriptome of the jakobid flagellate Reclinomonas americana), exploring the origins of the machinery responsible for the biogenesis of lysosome-related organelles (LROs), the Biogenesis of LRO Complexes (BLOCs 1,2, and 3). This pathway has been studied only in animals and is not considered a feature of the basic eukaryotic cell plan. We show that this machinery is present across the eukaryotic tree of life and was likely in place prior to LECA, making it an underappreciated facet of eukaryotic cellular organisation. Moreover, we resolve multiple points of ancient homology between all three BLOCs and other post-endosomal retrograde trafficking machinery (BORC, CCZ1 and MON1 proteins, and an unexpected relationship with the "homotypic fusion and vacuole protein sorting" (HOPS) and "Class C core vacuole/endosomal tethering" (CORVET) complexes), offering a mechanistic and evolutionary unification of these trafficking pathways. Overall, this study provides a comprehensive account of the rise of the LROs biogenesis machinery from before the LECA to current eukaryotic diversity, integrating it into the larger mechanistic framework describing endomembrane evolution.
Lysosomes are crucial in the tumour immune microenvironment, which is essential for the survival and homeostasis in multiple myeloma (MM). Here, we aimed to identify lysosome-related genes for the prognosis of MM and predicted their regulatory mechanisms. Gene expression profiles of MM from the GSE2658 and GSE57317 datasets were analysed. Lysosome-related differentially expressed genes (DEGs) were identified and used for molecular subtyping of MM patients. A prognostic model was constructed using univariate Cox regression and LASSO regression analyses. The relationship between prognostic genes, immune cell types, and autophagy pathways was assessed through correlation analysis. RT-qPCR was performed to validate the expression of prognostic genes in MM cells. A total of 9,954 DEGs were identified between high and low immune score groups, with 213 intersecting with lysosomal genes. Molecular subtyping revealed two distinct MM subtypes with significant differences in immune cell types and autophagy pathway activities. Five lysosome-related DEGs (CORO1A, ELANE, PSAP, RNASE2, and SNAPIN) were identified as significant prognostic markers. The prognostic model showed moderate predictive accuracy with AUC values up to 0.723. Prognostic genes demonstrated significant correlations with various immune cell types and autophagy pathways. Additionally, CORO1A, PSAP and RNASE2 expression was up-regulated in MM cells, while ELANE and SNAPIN were down-regulated. Five lysosomal genes in MM were identified, and a new risk model for prognosis was developed using these genes. This research could lead to discovering important gene markers for the treatment and prognosis of MM.
- MeSH
- autofagie genetika MeSH
- lidé MeSH
- lyzozomy * metabolismus genetika MeSH
- mnohočetný myelom * genetika imunologie MeSH
- nádorové biomarkery genetika MeSH
- nádorové mikroprostředí genetika imunologie MeSH
- prognóza MeSH
- regulace genové exprese u nádorů MeSH
- stanovení celkové genové exprese MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Despite the adenoids are regularly removed in patients with mucopolysaccharidoses (MPS), the underlying tissue and cellular pathologies remain understudied. We characterized an (immuno)histopathologic and ultrastructural phenotype dominated by lysosomal storage changes in a specific subset of adenotonsillar paracortical cells in 8 MPS patients (3 MPS I, 3 MPS II, and 2 MPS IIIA). These abnormal cells were effectively detected by an antibody targeting the lysosomal membrane tetraspanin CD63. Important, CD63+ storage vacuoles in these cells lacked the monocytes/macrophages lysosomal marker CD68. Such a distinct patterning of CD63 and CD68 was not present in a patient with infantile neurovisceral variant of acid sphingomyelinase deficiency. The CD63+ storage pathology was absent in two MPS I patients who either received enzyme-replacement therapy or underwent hematopoietic stem cells transplantation prior the adenoidectomy. Our study demonstrates novel features of lysosomal storage patterning and suggests diagnostic utility of CD63 detection in adenotonsillar lymphoid tissue of MPS patients.
- MeSH
- antigeny CD63 MeSH
- enzymová substituční terapie MeSH
- lidé MeSH
- lymfoidní tkáň patologie MeSH
- lyzozomy MeSH
- mukopolysacharidózy * diagnóza farmakoterapie genetika MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Truncating genetic variants of SORL1, encoding the endosome recycling receptor SORLA, have been accepted as causal of Alzheimer's disease (AD). However, most genetic variants observed in SORL1 are missense variants, for which it is complicated to determine the pathogenicity level because carriers come from pedigrees too small to be informative for penetrance estimations. Here, we describe three unrelated families in which the SORL1 coding missense variant rs772677709, that leads to a p.Y1816C substitution, segregates with Alzheimer's disease. Further, we investigate the effect of SORLA p.Y1816C on receptor maturation, cellular localization, and trafficking in cell-based assays. Under physiological circumstances, SORLA dimerizes within the endosome, allowing retromer-dependent trafficking from the endosome to the cell surface, where the luminal part is shed into the extracellular space (sSORLA). Our results showed that the p.Y1816C mutant impairs SORLA homodimerization in the endosome, leading to decreased trafficking to the cell surface and less sSORLA shedding. These trafficking defects of the mutant receptor can be rescued by the expression of the SORLA 3Fn-minireceptor. Finally, we find that iPSC-derived neurons with the engineered p.Y1816C mutation have enlarged endosomes, a defining cytopathology of AD. Our studies provide genetic as well as functional evidence that the SORL1 p.Y1816C variant is causal for AD. The partial penetrance of the mutation suggests this mutation should be considered in clinical genetic screening of multiplex early-onset AD families.
- MeSH
- Alzheimerova nemoc * genetika metabolismus patologie MeSH
- endozomy * metabolismus MeSH
- HEK293 buňky MeSH
- lidé středního věku MeSH
- lidé MeSH
- membránové transportní proteiny * genetika metabolismus MeSH
- missense mutace MeSH
- multimerizace proteinu MeSH
- proteiny související s LDL-receptory * genetika metabolismus MeSH
- rodokmen * MeSH
- senioři MeSH
- transport proteinů MeSH
- Check Tag
- lidé středního věku MeSH
- lidé MeSH
- mužské pohlaví MeSH
- senioři MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
Lipid nanoparticle (LNP)-mRNA complexes are transforming medicine. However, the medical applications of LNPs are limited by their low endosomal disruption rates, high toxicity and long tissue persistence times. LNPs that rapidly hydrolyse in endosomes (RD-LNPs) could solve the problems limiting LNP-based therapeutics and dramatically expand their applications but have been challenging to synthesize. Here we present an acid-degradable linker termed 'azido-acetal' that hydrolyses in endosomes within minutes and enables the production of RD-LNPs. Acid-degradable lipids composed of polyethylene glycol lipids, anionic lipids and cationic lipids were synthesized with the azido-acetal linker and used to generate RD-LNPs, which significantly improved the performance of LNP-mRNA complexes in vitro and in vivo. Collectively, RD-LNPs delivered mRNA more efficiently to the liver, lung, spleen and brains of mice and to haematopoietic stem and progenitor cells in vitro than conventional LNPs. These experiments demonstrate that engineering LNP hydrolysis rates in vivo has great potential for expanding the medical applications of LNPs.
The mechanistic target of rapamycin complex 1 (mTORC1) pathway regulates cell growth and metabolism in response to many environmental cues, including nutrients. Amino acids signal to mTORC1 by modulating the guanine nucleotide loading states of the heterodimeric Rag GTPases, which bind and recruit mTORC1 to the lysosomal surface, its site of activation. The Rag GTPases are tethered to the lysosome by the Ragulator complex and regulated by the GATOR1, GATOR2, and KICSTOR multiprotein complexes that localize to the lysosomal surface through an unknown mechanism(s). Here, we show that mTORC1 is completely insensitive to amino acids in cells lacking the Rag GTPases or the Ragulator component p18. Moreover, not only are the Rag GTPases and Ragulator required for amino acids to regulate mTORC1, they are also essential for the lysosomal recruitment of the GATOR1, GATOR2, and KICSTOR complexes, which stably associate and traffic to the lysosome as the "GATOR" supercomplex. The nucleotide state of RagA/B controls the lysosomal association of GATOR, in a fashion competitively antagonized by the N terminus of the amino acid transporter SLC38A9. Targeting of Ragulator to the surface of mitochondria is sufficient to relocalize the Rags and GATOR to this organelle, but not to enable the nutrient-regulated recruitment of mTORC1 to mitochondria. Thus, our results reveal that the Rag-Ragulator complex is the central organizer of the physical architecture of the mTORC1 nutrient-sensing pathway and underscore that mTORC1 activation requires signal transduction on the lysosomal surface.
- MeSH
- adaptorové proteiny signální transdukční metabolismus MeSH
- aminokyseliny * metabolismus MeSH
- HEK293 buňky MeSH
- lidé MeSH
- lyzozomy * metabolismus MeSH
- mechanistické cílové místo rapamycinového komplexu 1 * metabolismus MeSH
- monomerní proteiny vázající GTP * metabolismus MeSH
- myši MeSH
- signální transdukce * MeSH
- živiny * metabolismus MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Močové extracelulární vezikuly (uEVs) představují slibný nástroj pro neinvazivní diagnostiku a monitorování onemocnění ledvin. Tyto vezikuly, secernované buňkami ledvin, obsahují biomolekuly odrážející stav mateřských buněk. Výzkum se zaměřuje na využití uEVs jako biomarkerů pro chronická onemocnění ledvin, akutní poškození ledvin, diabetickou nefropatii a monitorování po transplantaci ledvin. Kromě diagnostického potenciálu jsou uEVs zkoumány pro terapeutické aplikace v regeneraci ledvinové tkáně. Přes výzvy v standardizaci izolačních a analytických metod uEVs, pokrok ve vývoji metod charakterizace uEVs podporuje jejich klinické využití.
Urinary extracellular vesicles (uEVs) represent a promising tool for non-invasive diagnosis and monitoring of kidney disease. These vesicles, secreted by kidney cells, contain biomolecules reflecting the status of the parent cells. Research focuses on using uEVs as biomarkers for chronic kidney disease, acute kidney injury, diabetic nephropathy and monitoring kidney transplantation. In addition to diagnostic potential, uEVs are being investigated for therapeutic applications in kidney tissue regeneration. Despite challenges in standardizing uEVs isolation and analysis methods, progress in the development of uEVs characterization methods supports their clinical use.
- Klíčová slova
- močové extracelulární vezikuly,
- MeSH
- biologické markery * analýza MeSH
- COP-vezikuly fyziologie MeSH
- lidé MeSH
- nemoci ledvin * diagnóza patofyziologie MeSH
- urogenitální systém anatomie a histologie MeSH
- Check Tag
- lidé MeSH
