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Keywords = extracellular cathepsin D

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13 pages, 2537 KiB  
Article
Molecular Insights into the Interaction of Cathepsin D and Iron in Chronic Wound Healing: Exploring Therapeutic Potential and Mechanisms
by María Rodríguez-Moreno and Isabel Legaz
Biomedicines 2025, 13(3), 544; https://doi.org/10.3390/biomedicines13030544 - 21 Feb 2025
Viewed by 976
Abstract
Background: Chronic wounds, such as diabetic ulcers, often fail to progress through healing due to persistent inflammation, infections, and extracellular matrix (ECM) imbalances. Cathepsin D, an aspartate protease active in acidic environments, plays a pivotal role in wound healing by mediating inflammatory responses, [...] Read more.
Background: Chronic wounds, such as diabetic ulcers, often fail to progress through healing due to persistent inflammation, infections, and extracellular matrix (ECM) imbalances. Cathepsin D, an aspartate protease active in acidic environments, plays a pivotal role in wound healing by mediating inflammatory responses, ECM remodeling, and macrophage phenotype transitions. Its dysregulation, however, can impair healing, highlighting the need for targeted modulation of its activity. The aim of this study was to investigate the molecular interaction between Fe2+ and cathepsin D’s catalytic core and ionic zipper under physiological and acidic conditions to identify strategies to enhance tissue repair and accelerate the healing of chronic wounds. Methods: The molecular structure of active cathepsin D was obtained from the Protein Data Bank (PDB) and analyzed using UCSF Chimera. Molecular interactions between cathepsin D and ferrous ions (Fe2+) were studied, focusing on key residues (D33 and D231) and ionic zipper residues (E5, E180, and D187). Results: Our results showed that the active form of cathepsin D, a 96 kDa dimer, consisted of heterodimers with distinct amino acid chains, where residues D33 and D231 formed the active site, and E5, E180, and D187 constituted the ionic zipper. A functional pocket containing the conserved residues D33 and D231, essential for proteolytic activity, was identified. At physiological pH (~7.5), D33 exhibited the most potent interactions with Fe2+, with interaction energies of −7 × 1017 J at oxygen atoms of the carboxylate group (OD1) and α-carbon (CA) atoms, whereas D231 showed slightly lower energies of −6 × 1017 J at γ-carbon atom (CG) and CA atoms. At acidic pH (~4), E5 was the primary interacting residue, with the shortest distance to Fe2+ (2.69 Å), and showed stable interactions across several atoms, emphasizing its role in metal binding. Conclusions: pH conditions strongly influence the interaction of cathepsin D with Fe2. At physiological pH, residues D33 and D231 demonstrate robust and energetically efficient binding with Fe2+. At the same time, under acidic conditions, E5 emerges as the primary residue involved, potentially affecting the ionic zipper of cathepsin D. These insights provide a molecular foundation for targeting specific residues to modulate cathepsin D activity, presenting promising opportunities for therapeutic strategies aimed at improving chronic wound healing. Full article
(This article belongs to the Special Issue Wound Healing: From Mechanisms to Therapeutic Approaches)
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27 pages, 8155 KiB  
Article
Secretome Analysis of Prostate Cancer Cell Lines Reveals Cell Cycle-Dependent PSA Secretion and Potential Biomarkers
by Eshwari Dathathri, Yvette Peters, Diana Andreoli, Mel Bruins, Jaco Kraan, Leon W. M. M. Terstappen and Ruchi Bansal
Cancers 2025, 17(5), 721; https://doi.org/10.3390/cancers17050721 - 20 Feb 2025
Cited by 1 | Viewed by 1312
Abstract
Background: Metastatic prostate cancer (mPCa) is marked by heterogeneity and therapy resistance, which arise from prolonged therapy regimens. This heterogeneity is reflected in various morphologic and genetic characteristics, biomarker expression, and other molecular mechanisms, thereby contributing to the complexity of the disease. Methods: [...] Read more.
Background: Metastatic prostate cancer (mPCa) is marked by heterogeneity and therapy resistance, which arise from prolonged therapy regimens. This heterogeneity is reflected in various morphologic and genetic characteristics, biomarker expression, and other molecular mechanisms, thereby contributing to the complexity of the disease. Methods: To investigate tumor heterogeneity, the effects of androgen targeting therapy (ADT) on single-cell PSA secretion was assessed by analyzing the prostate cancer cell lines using a modified ELISpot platform. The FACS and cytospin techniques were employed to understand the influence of the cell cycle on PSA secretion patterns. Additionally, a proteome array was used to identify potential biomarkers from different PCa cell lines with varying metastatic potential. Results: Among the various PCa cell lines examined, PSA expression and secretion could be visualized only from the LNCaPs. PSA secretion from circulating tumor cells (CTCs) further confirmed the validity of this assay. These LNCaPs exhibited heterogeneity in single-cell intracellular and extracellular PSA expression and in their ADT responses. LNCaPs in the G1 phase showed higher PSA secretion than in the S or G2/M phase. Apart from PSA, Cathepsin D, Progranulin, IL-8, Serpin E1, and Enolase 2 were identified as secretome markers from the metastatic PCa cell lines. Conclusions: We observed variability in PSA secretion in LNCaP in response to anti-androgen treatment and a cell cycle-dependent secretion pattern. The notable presence of Progranulin and Cathepsin D in metastatic cell lines makes them promising candidates for use in multiplexing and single-cell platforms, potentially advancing our understanding and treatment of this disease. Full article
(This article belongs to the Special Issue Clinical Treatment and Prognostic Factors of Urologic Cancer)
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13 pages, 6278 KiB  
Article
Avellanin A Has an Antiproliferative Effect on TP-Induced RWPE-1 Cells via the PI3K-Akt Signalling Pathway
by Chang Xu, Guangping Cao, Hong Zhang, Meng Bai, Xiangxi Yi and Xinjian Qu
Mar. Drugs 2024, 22(6), 275; https://doi.org/10.3390/md22060275 - 13 Jun 2024
Cited by 4 | Viewed by 1893
Abstract
Cyclic pentapeptide compounds have garnered much attention as a drug discovery resource. This study focused on the characterization and anti-benign prostatic hyperplasia (BPH) properties of avellanin A from Aspergillus fumigatus fungus in marine sediment samples collected in the Beibu Gulf of Guangxi Province [...] Read more.
Cyclic pentapeptide compounds have garnered much attention as a drug discovery resource. This study focused on the characterization and anti-benign prostatic hyperplasia (BPH) properties of avellanin A from Aspergillus fumigatus fungus in marine sediment samples collected in the Beibu Gulf of Guangxi Province in China. The antiproliferative effect and molecular mechanism of avellanin A were explored in testosterone propionate (TP)-induced RWPE-1 cells. The transcriptome results showed that avellanin A significantly blocked the ECM–receptor interaction and suppressed the downstream PI3K-Akt signalling pathway. Molecular docking revealed that avellanin A has a good affinity for the cathepsin L protein, which is involved in the terminal degradation of extracellular matrix components. Subsequently, qRT-PCR analysis revealed that the expression of the genes COL1A1, COL1A2, COL5A2, COL6A3, MMP2, MMP9, ITGA2, and ITGB3 was significantly downregulated after avellanin A intervention. The Western blot results also confirmed that it not only reduced ITGB3 and FAK/p-FAK protein expression but also inhibited PI3K/p-PI3K and Akt/p-Akt protein expression in the PI3K-Akt signalling pathway. Furthermore, avellanin A downregulated Cyclin D1 protein expression and upregulated Bax, p21WAF1/Cip1, and p53 proapoptotic protein expression in TP-induced RWPE-1 cells, leading to cell cycle arrest and inhibition of cell proliferation. The results of this study support the use of avellanin A as a potential new drug for the treatment of BPH. Full article
(This article belongs to the Special Issue Pharmacological Potential of Marine Natural Products)
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11 pages, 3015 KiB  
Article
Involvement of Endolysosomes and Aurora Kinase A in the Regulation of Amyloid β Protein Levels in Neurons
by Zahra Afghah, Nabab Khan, Gaurav Datta, Peter W. Halcrow, Jonathan D. Geiger and Xuesong Chen
Int. J. Mol. Sci. 2024, 25(11), 6200; https://doi.org/10.3390/ijms25116200 - 4 Jun 2024
Cited by 3 | Viewed by 1467
Abstract
Aurora kinase A (AURKA) is a serine/threonine-protein kinase that regulates microtubule organization during neuron migration and neurite formation. Decreased activity of AURKA was found in Alzheimer’s disease (AD) brain samples, but little is known about the role of AURKA in AD pathogenesis. Here, [...] Read more.
Aurora kinase A (AURKA) is a serine/threonine-protein kinase that regulates microtubule organization during neuron migration and neurite formation. Decreased activity of AURKA was found in Alzheimer’s disease (AD) brain samples, but little is known about the role of AURKA in AD pathogenesis. Here, we demonstrate that AURKA is expressed in primary cultured rat neurons, neurons from adult mouse brains, and neurons in postmortem human AD brains. AURKA phosphorylation, which positively correlates with its activity, is reduced in human AD brains. In SH-SY5Y cells, pharmacological activation of AURKA increased AURKA phosphorylation, acidified endolysosomes, decreased the activity of amyloid beta protein (Aβ) generating enzyme β-site amyloid precursor protein cleaving enzyme (BACE-1), increased the activity of the Aβ degrading enzyme cathepsin D, and decreased the intracellular and secreted levels of Aβ. Conversely, pharmacological inhibition of AURKA decreased AURKA phosphorylation, de-acidified endolysosomes, decreased the activity of cathepsin D, and increased intracellular and secreted levels of Aβ. Thus, reduced AURKA activity in AD may contribute to the development of intraneuronal accumulations of Aβ and extracellular amyloid plaque formation. Full article
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11 pages, 1630 KiB  
Communication
A New Cathepsin D Targeting Drug Delivery System Based on Immunoliposomes Functionalized with Lipidated Pepstatin A
by Andreja Kozak, Georgy Mikhaylov, Pavlo Khodakivskyi, Elena Goun, Boris Turk and Olga Vasiljeva
Pharmaceutics 2023, 15(10), 2464; https://doi.org/10.3390/pharmaceutics15102464 - 14 Oct 2023
Cited by 4 | Viewed by 2000
Abstract
Cathepsin D is an aspartic protease and one of the most abundant proteases. It is overexpressed in many cancers and plays an important role in tumor development, progression, and metastasis. While it is a physiologically intracellular protein, cathepsin D is secreted into the [...] Read more.
Cathepsin D is an aspartic protease and one of the most abundant proteases. It is overexpressed in many cancers and plays an important role in tumor development, progression, and metastasis. While it is a physiologically intracellular protein, cathepsin D is secreted into the extracellular matrix under pathological conditions, making it an appealing target for drug delivery systems. Here, we present the development and evaluation of a new delivery system for tumor targeting based on immunoliposomes functionalized with pepstatin A—a natural peptide inhibitor of cathepsin D. A lipid tail was added to pepstatin A, enabling its incorporation into the liposomal lipid bilayer. The successful targeting of cathepsin D was confirmed using recombinant cathepsin D and in tumor cell lines, showing the feasibility of this targeting approach and its potential for in vivo use in theragnostic applications. Full article
(This article belongs to the Special Issue Targeted Drug Delivery to Improve Cancer Therapy)
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20 pages, 3045 KiB  
Article
Sputum from People with Cystic Fibrosis Reduces the Killing of Methicillin-Resistant Staphylococcus aureus by Neutrophils and Diminishes Phagosomal Production of Reactive Oxygen Species
by Kayla M. Fantone, Joanna B. Goldberg, Arlene A. Stecenko and Balázs Rada
Pathogens 2023, 12(9), 1148; https://doi.org/10.3390/pathogens12091148 - 9 Sep 2023
Cited by 3 | Viewed by 1886
Abstract
Cystic fibrosis (CF) airway disease is characterized by chronic polymicrobial infections and an infiltration of neutrophils (PMNs). Staphylococcus aureus has been the most prevalent respiratory pathogen in CF. In particular, methicillin-resistant S. aureus (MRSA) represents a huge clinical burden in CF due to [...] Read more.
Cystic fibrosis (CF) airway disease is characterized by chronic polymicrobial infections and an infiltration of neutrophils (PMNs). Staphylococcus aureus has been the most prevalent respiratory pathogen in CF. In particular, methicillin-resistant S. aureus (MRSA) represents a huge clinical burden in CF due to its association with lung disease and increased resistance to antibiotics. In CF, PMNs are unable to kill and clear MRSA. The reason for this remains largely unknown. Our study found that CF PMNs are as equally capable of killing MRSA as healthy PMNs. We show that the CF sputum, however, significantly impairs the ability of human PMNs to kill CF MRSA isolates. In the absence of CF sputum, PMNs kill MRSA via intracellular mechanisms mediated by phagocytosis, rather than extracellular mechanisms via NET formation. CF sputum does not affect the phagocytosis of MRSA via healthy or CF PMNs. Our results demonstrate that CF sputum exposure impairs phagosomal levels of reactive oxygen species (ROS) in MRSA-phagocytosing PMNs. While phagosomal co-localizations of MRSA with primary granule markers, myeloperoxidase and cathepsin D, were significantly reduced upon CF sputum exposure, that of a third azurophilic granule marker, neutrophil elastase, remained unaffected. This suggests that CF sputum does not compromise the fusion of primary granules with phagosomes but diminishes phagosomal ROS levels via another, likely more specific, mechanism. Overall, we identified the airway environment as an important factor that restricts neutrophils’ oxidative microbicidal activities in CF against MRSA. These results deliver new details of the complex host–pathogen interactions present in the CF lung. Full article
(This article belongs to the Section Immunological Responses and Immune Defense Mechanisms)
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19 pages, 6396 KiB  
Article
Impact of Enniatin B and Beauvericin on Lysosomal Cathepsin B Secretion and Apoptosis Induction
by Mohammed Aufy, Ramadan F. Abdelaziz, Ahmed M. Hussein, Nermina Topcagic, Hadil Shamroukh, Mostafa A. Abdel-Maksoud, Tamer Z. Salem and Christian R. Studenik
Int. J. Mol. Sci. 2023, 24(3), 2030; https://doi.org/10.3390/ijms24032030 - 19 Jan 2023
Cited by 14 | Viewed by 7477
Abstract
Enniatin B (ENN B) and Beauvericin (BEA) are cyclohexadepsipeptides that can be isolated from Fusarium and Beauveria bassiana, respectively. Both compounds are cytotoxic and ionophoric. In the present study, the mechanism of cell death induced by these compounds was investigated. Epidermal carcinoma-derived [...] Read more.
Enniatin B (ENN B) and Beauvericin (BEA) are cyclohexadepsipeptides that can be isolated from Fusarium and Beauveria bassiana, respectively. Both compounds are cytotoxic and ionophoric. In the present study, the mechanism of cell death induced by these compounds was investigated. Epidermal carcinoma-derived cell line KB-3-1 cells were treated with different concentrations of these compounds. The extracellular secretion of cathepsin B increased in a concentration-dependent manner, and the lysosomal staining by lysotracker red was reduced upon the treatment with any of the compounds. However, the extracellular secretion of cathepsin L and cathepsin D were not affected. Inhibition of cathepsin B with specific inhibitor CA074 significantly reduced the cytotoxic effect of both compounds, while inhibition of cathepsin D or cathepsin L did not influence the cytotoxic activities of both compounds. In vitro labelling of lysosomal cysteine cathepsins with Ethyl (2S, 3S)-epoxysuccinate-Leu-Tyr-Acp-Lys (Biotin)-NH2 (DCG04) was not affected in case of cathepsin L upon the treatment with both compounds, while it was significantly reduced in case of cathepsin B. In conclusion, ENN B and BEA increase lysosomal Ph, which inhibits delivery of cathepsin B from Golgi to lysosomes, thereby inducing cathepsin B release in cytosol, which activates caspases and hence the apoptotic pathway. Full article
(This article belongs to the Special Issue Cell Apoptosis 2.0)
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13 pages, 1844 KiB  
Article
Plasma Small Extracellular Vesicle Cathepsin D Dysregulation in GRN/C9orf72 and Sporadic Frontotemporal Lobar Degeneration
by Sonia Bellini, Claudia Saraceno, Luisa Benussi, Andrea Geviti, Antonio Longobardi, Roland Nicsanu, Sara Cimini, Martina Ricci, Laura Canafoglia, Cinzia Coppola, Gianfranco Puoti, Giuliano Binetti, Giacomina Rossi and Roberta Ghidoni
Int. J. Mol. Sci. 2022, 23(18), 10693; https://doi.org/10.3390/ijms231810693 - 14 Sep 2022
Cited by 5 | Viewed by 2905
Abstract
Emerging data suggest the roles of endo-lysosomal dysfunctions in frontotemporal lobar degeneration (FTLD) and in other dementias. Cathepsin D is one of the major lysosomal proteases, mediating the degradation of unfolded protein aggregates. In this retrospective study, we investigated cathepsin D levels in [...] Read more.
Emerging data suggest the roles of endo-lysosomal dysfunctions in frontotemporal lobar degeneration (FTLD) and in other dementias. Cathepsin D is one of the major lysosomal proteases, mediating the degradation of unfolded protein aggregates. In this retrospective study, we investigated cathepsin D levels in human plasma and in the plasma small extracellular vesicles (sEVs) of 161 subjects (40 sporadic FTLD, 33 intermediate/pathological C9orf72 expansion carriers, 45 heterozygous/homozygous GRN mutation carriers, and 43 controls). Cathepsin D was quantified by ELISA, and nanoparticle tracking analysis data (sEV concentration for the cathepsin D level normalization) were extracted from our previously published dataset or were newly generated. First, we revealed a positive correlation of the cathepsin D levels with the age of the patients and controls. Even if no significant differences were found in the cathepsin D plasma levels, we observed a progressive reduction in plasma cathepsin D moving from the intermediate to C9orf72 pathological expansion carriers. Observing the sEVs nano-compartment, we observed increased cathepsin D sEV cargo (ng/sEV) levels in genetic/sporadic FTLD. The diagnostic performance of this biomarker was fairly high (AUC = 0.85). Moreover, sEV and plasma cathepsin D levels were positively correlated with age at onset. In conclusion, our study further emphasizes the common occurrence of endo-lysosomal dysregulation in GRN/C9orf72 and sporadic FTLD. Full article
(This article belongs to the Special Issue Exosomes)
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16 pages, 3376 KiB  
Article
Functional Analysis of the Cathepsin D Gene Response to SGIV Infection in the Orange-Spotted Grouper, Epinephelus coioides
by Yuexuan Wang, Honglin Han, Kecheng Zhu, Suifeng Xu, Chengzong Han, Yunxiang Jiang, Shina Wei and Qiwei Qin
Viruses 2022, 14(8), 1680; https://doi.org/10.3390/v14081680 - 29 Jul 2022
Cited by 3 | Viewed by 2325
Abstract
(1) Background: Lysosomal aspartic protease Cathepsin D (CD) is a key regulator and signaling molecule in various biological processes including activation and degradation of intracellular proteins, the antigen process and programmed cell death. However, the function of fish CD in virus infection remains [...] Read more.
(1) Background: Lysosomal aspartic protease Cathepsin D (CD) is a key regulator and signaling molecule in various biological processes including activation and degradation of intracellular proteins, the antigen process and programmed cell death. However, the function of fish CD in virus infection remains largely unknown. (2) Methods: The functions of the CD gene response to SGIV infection was determined with light microscopy, reverse transcription quantitative PCR, Western blot and flow cytometry. (3) Results: In this study, Ec-Cathepsin D (Ec-CD) was cloned and identified from the orange-spotted grouper, Epinephelus coioides. The open reading frame (ORF) of Ec-CD consisted of 1191 nucleotides encoding a 396 amino acid protein with a predicted molecular mass of 43.17 kDa. Ec-CD possessed typical CD structural features including an N-terminal signal peptide, a propeptide region and a mature domain including two glycosylation sites and two active sites, which were conserved in other CD sequences. Ec-CD was predominantly expressed in the spleen and kidneys of healthy groupers. A subcellular localization assay indicated that Ec-CD was mainly distributed in the cytoplasm. Ec-CD expression was suppressed by SGIV stimulation and Ec-CD-overexpressing inhibited SGIV replication, SGIV-induced apoptosis, caspase 3/8/9 activity and the activation of reporter gene p53 and activating protein-1 (AP-1) in vitro. Simultaneously, Ec-CD overexpression obviously restrained the activated mitogen-activated protein kinase (MAPK) pathways, including extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK). In addition, Ec-CD overexpression negatively regulated the transcription level of pro-inflammatory cytokines and activation of the NF-κB promotor. (4) Conclusions: Our findings revealed that the Ec-CD possibly served a function during SGIV infection. Full article
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19 pages, 3611 KiB  
Article
Mechanistic Effects of Baicalein on Aqueous Humor Drainage and Intraocular Pressure
by Hoi-lam Li, Sze Wan Shan, W. Daniel Stamer, King-kit Li, Henry Ho-lung Chan, Mortimer M. Civan, Chi-ho To, Thomas Chuen Lam and Chi-wai Do
Int. J. Mol. Sci. 2022, 23(13), 7372; https://doi.org/10.3390/ijms23137372 - 1 Jul 2022
Cited by 5 | Viewed by 3214
Abstract
Elevated intraocular pressure (IOP) is a major risk factor for glaucoma that results from impeded fluid drainage. The increase in outflow resistance is caused by trabecular meshwork (TM) cell dysfunction and excessive extracellular matrix (ECM) deposition. Baicalein (Ba) is a natural flavonoid and [...] Read more.
Elevated intraocular pressure (IOP) is a major risk factor for glaucoma that results from impeded fluid drainage. The increase in outflow resistance is caused by trabecular meshwork (TM) cell dysfunction and excessive extracellular matrix (ECM) deposition. Baicalein (Ba) is a natural flavonoid and has been shown to regulate cell contraction, fluid secretion, and ECM remodeling in various cell types, suggesting the potential significance of regulating outflow resistance and IOP. We demonstrated that Ba significantly lowered the IOP by about 5 mmHg in living mice. Consistent with that, Ba increased the outflow facility by up to 90% in enucleated mouse eyes. The effects of Ba on cell volume regulation and contractility were examined in primary human TM (hTM) cells. We found that Ba (1–100 µM) had no effect on cell volume under iso-osmotic conditions but inhibited the regulatory volume decrease (RVD) by up to 70% under hypotonic challenge. In addition, Ba relaxed hTM cells via reduced myosin light chain (MLC) phosphorylation. Using iTRAQ-based quantitative proteomics, 47 proteins were significantly regulated in hTM cells after a 3-h Ba treatment. Ba significantly increased the expression of cathepsin B by 1.51-fold and downregulated the expression of D-dopachrome decarboxylase and pre-B-cell leukemia transcription factor-interacting protein 1 with a fold-change of 0.58 and 0.40, respectively. We suggest that a Ba-mediated increase in outflow facility is triggered by cell relaxation via MLC phosphorylation along with inhibiting RVD in hTM cells. The Ba-mediated changes in protein expression support the notion of altered ECM homeostasis, potentially contributing to a reduction of outflow resistance and thereby IOP. Full article
(This article belongs to the Special Issue Proteomics and Its Applications in Disease)
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19 pages, 6419 KiB  
Article
Low Concentration of the Neutrophil Proteases Cathepsin G, Cathepsin B, Proteinase-3 and Metalloproteinase-9 Induce Biofilm Formation in Non-Biofilm-Forming Staphylococcus epidermidis Isolates
by Itzia S. Gómez-Alonso, Sergio Martínez-García, Gabriel Betanzos-Cabrera, Esmeralda Juárez, María C. Sarabia-León, María Teresa Herrera, Fernando Gómez-Chávez, Luvia Sanchez-Torres, Sandra Rodríguez-Martínez, Mario E. Cancino-Diaz, Jorge Cancino and Juan C. Cancino-Diaz
Int. J. Mol. Sci. 2022, 23(9), 4992; https://doi.org/10.3390/ijms23094992 - 30 Apr 2022
Cited by 4 | Viewed by 2958
Abstract
Neutrophils play a crucial role in eliminating bacteria that invade the human body; however, cathepsin G can induce biofilm formation in a non-biofilm-forming Staphylococcus epidermidis 1457 strain, suggesting that neutrophil proteases may be involved in biofilm formation. Cathepsin G, cathepsin B, proteinase-3, and [...] Read more.
Neutrophils play a crucial role in eliminating bacteria that invade the human body; however, cathepsin G can induce biofilm formation in a non-biofilm-forming Staphylococcus epidermidis 1457 strain, suggesting that neutrophil proteases may be involved in biofilm formation. Cathepsin G, cathepsin B, proteinase-3, and metalloproteinase-9 (MMP-9) from neutrophils were tested on the biofilm induction in commensal (skin isolated) and clinical non-biofilm-forming S. epidermidis isolates. From 81 isolates, 53 (74%) were aap+, icaA, icaD genotype, and without the capacity of biofilm formation under conditions of 1% glucose, 4% ethanol or 4% NaCl, but these 53 non-biofilm-forming isolates induced biofilm by the use of different neutrophil proteases. Of these, 62.3% induced biofilm with proteinase-3, 15% with cathepsin G, 10% with cathepsin B and 5% with MMP -9, where most of the protease-induced biofilm isolates were commensal strains (skin). In the biofilm formation kinetics analysis, the addition of phenylmethylsulfonyl fluoride (PMSF; a proteinase-3 inhibitor) showed that proteinase-3 participates in the cell aggregation stage of biofilm formation. A biofilm induced with proteinase-3 and DNAse-treated significantly reduced biofilm formation at an early time (initial adhesion stage of biofilm formation) compared to untreated proteinase-3-induced biofilm (p < 0.05). A catheter inoculated with a commensal (skin) non-biofilm-forming S. epidermidis isolate treated with proteinase-3 and another one without the enzyme were inserted into the back of a mouse. After 7 days of incubation period, the catheters were recovered and the number of grown bacteria was quantified, finding a higher amount of adhered proteinase-3-treated bacteria in the catheter than non-proteinase-3-treated bacteria (p < 0.05). Commensal non-biofilm-forming S. epidermidis in the presence of neutrophil cells significantly induced the biofilm formation when multiplicity of infection (MOI) 1:0.01 (neutrophil:bacteria) was used, but the addition of a cocktail of protease inhibitors impeded biofilm formation. A neutrophil:bacteria assay did not induce neutrophil extracellular traps (NETs). Our results suggest that neutrophils, in the presence of commensal non-biofilm-forming S. epidermidis, do not generate NETs formation. The effect of neutrophils is the production of proteases, and proteinase-3 releases bacterial DNA at the initial adhesion, favoring cell aggregation and subsequently leading to biofilm formation. Full article
(This article belongs to the Special Issue Neutrophil in Cell Biology and Diseases)
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16 pages, 3811 KiB  
Article
The Different Effect of Decellularized Myocardial Matrix Hydrogel and Decellularized Small Intestinal Submucosa Matrix Hydrogel on Cardiomyocytes and Ischemic Heart
by Xifeng Yang, Shihao Chen, Jiaxin Chen, Yunqi Liu, Ying Bai, Shengli Yin and Daping Quan
Appl. Sci. 2021, 11(17), 7768; https://doi.org/10.3390/app11177768 - 24 Aug 2021
Cited by 8 | Viewed by 2734
Abstract
Injectable decellularized matrix hydrogels derived from either myocardium or small intestinal submucosa (pDMYO-gel, pDSIS-gel) have been successfully used for myocardial injury repair. However, the relationship between tissue-specific biological functions and protein composition in these two materials is not clear yet. In this study, [...] Read more.
Injectable decellularized matrix hydrogels derived from either myocardium or small intestinal submucosa (pDMYO-gel, pDSIS-gel) have been successfully used for myocardial injury repair. However, the relationship between tissue-specific biological functions and protein composition in these two materials is not clear yet. In this study, the protein composition, mechanical properties, and morphology of these two hydrogels and their effects on the behavior of neonatal rat cardiomyocytes (NRCMs) and human umbilical vein endothelial cells (HUVECs), are investigated. The results show that pDMYO-gel is more conducive to growth, adhesion, spreading, and maintenance of normal NRCM beating, due to its higher proportion of extracellular matrix (ECM) glycoproteins (49.55%) and some unique functional proteins such as annexin-6 (ANXA6), agrin (AGRN), cathepsin D (CTSD) and galectin-1 (LGALS1), whereas pDSIS-gel is more conducive to the proliferation of HUVECs. Animal study shows that pDMYO-gel has a better effect on improving cardiac function, inhibiting myocardial fibrosis and maintaining ventricular wall thickness in acute myocardial infarction models in vivo. Therefore, it is proposed that injectable pDMYO-gel hydrogel may be more suitable for functional recovery of myocardial injuries. Full article
(This article belongs to the Section Applied Biosciences and Bioengineering)
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16 pages, 8008 KiB  
Article
Release of Iron-Loaded Ferritin in Sodium Iodate-Induced Model of Age Related Macular Degeneration: An In-Vitro and In-Vivo Study
by Ajay Ashok, Suman Chaudhary, Aaron S. Wise, Neil A. Rana, Dallas McDonald, Alexander E. Kritikos, Ewald Lindner and Neena Singh
Antioxidants 2021, 10(8), 1253; https://doi.org/10.3390/antiox10081253 - 5 Aug 2021
Cited by 10 | Viewed by 3597
Abstract
In this report, we evaluated the role of iron in sodium iodate (NaIO3)-induced model of age-related macular degeneration (AMD) in ARPE-19 cells in-vitro, and mouse models in-vivo. ARPE-19 cells, a human retinal pigmented epithelial cell line, were exposed to 10 mM [...] Read more.
In this report, we evaluated the role of iron in sodium iodate (NaIO3)-induced model of age-related macular degeneration (AMD) in ARPE-19 cells in-vitro, and mouse models in-vivo. ARPE-19 cells, a human retinal pigmented epithelial cell line, were exposed to 10 mM of NaIO3 for 24 h, and the expression and localization of major iron modulating proteins was evaluated by Western blotting (WB) and immunostaining. Synthesis and maturation of cathepsin-D (cat-D), a lysosomal enzyme, was evaluated by quantitative reverse-transcriptase polymerase chain reaction (RT-qPCR) and WB respectively. For in-vivo studies, C57BL/6 mice were injected with 40 mg/kg mouse body weight of NaIO3 intraperitoneally, and their retina was evaluated after 3 weeks as above. We observed that NaIO3 induced a 10-fold increase in ferritin in ARPE-19 cells, which co-localized with LC3II, an autophagosomal marker, and LAMP-1, a lysosomal marker. A similar increase in ferritin was noted in retinal lysates and retinal sections of NaIO3-injected mice by WB and immunostaining. Impaired synthesis and maturation of cat-D was also noted. Accumulated ferritin was loaded with iron, and released from retinal pigmented epithelial (RPE) cells in Perls’ and LAMP-1 positive vesicles. These observations suggest that NaIO3 impairs lysosomal degradation of ferritin by decreasing the transcription and maturation of cat-D in RPE-19 cells. Iron-loaded ferritin accumulates in lysosomes and is released in lysosome membrane-enclosed vesicles in the extracellular milieu. Accumulation of ferritin in RPE-19 cells and fusion of ferritin-containing vesicles with adjacent photoreceptor cells is likely to create iron overload, compromising their viability. Moreover, reduced activity of cat-D is likely to promote the accumulation of other cellular debris in lysosomal vesicles, contributing to AMD-like pathology. Full article
(This article belongs to the Special Issue Oxidative Stress-Induced Eye Diseases and Therapeutic Interventions)
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17 pages, 12680 KiB  
Article
Simulated Microgravity Remodels Extracellular Matrix of Osteocommitted Mesenchymal Stromal Cells
by Ivan Zhivodernikov, Andrey Ratushnyy and Ludmila Buravkova
Int. J. Mol. Sci. 2021, 22(11), 5428; https://doi.org/10.3390/ijms22115428 - 21 May 2021
Cited by 16 | Viewed by 3301
Abstract
The extracellular matrix (ECM) is the principal structure of bone tissue. Long-term spaceflights lead to osteopenia, which may be a result of the changes in composition as well as remodeling of the ECM by osteogenic cells. To elucidate the cellular effects of microgravity, [...] Read more.
The extracellular matrix (ECM) is the principal structure of bone tissue. Long-term spaceflights lead to osteopenia, which may be a result of the changes in composition as well as remodeling of the ECM by osteogenic cells. To elucidate the cellular effects of microgravity, human mesenchymal stromal cells (MSCs) and their osteocommitted progeny were exposed to simulated microgravity (SMG) for 10 days using random positioning machine (RPM). After RPM exposure, an imbalance of MSC collagen/non-collagen ratio at the expense of a decreased level of collagenous proteins was detected. At the same time, the secretion of proteases (cathepsin A, cathepsin D, MMP3) was increased. No significant effects of SMG on the expression of stromal markers and cell adhesion molecules on the MSC surface were noted. Upregulation of COL11A1, CTNND1, TIMP3, and TNC and downregulation of HAS1, ITGA3, ITGB1, LAMA3, MMP1, and MMP11 were detected in RPM exposed MSCs. ECM-associated transcriptomic changes were more pronounced in osteocommitted progeny. Thus, 10 days of SMG provokes a decrease in the collagenous components of ECM, probably due to the decrease in collagen synthesis and activation of proteases. The presented data demonstrate that ECM-associated molecules of both native and osteocommitted MSCs may be involved in bone matrix reorganization during spaceflight. Full article
(This article belongs to the Special Issue Microgravity and Space Medicine 2.0)
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8 pages, 6572 KiB  
Article
Internalization of Clostridium botulinum C2 Toxin Is Regulated by Cathepsin B Released from Lysosomes
by Masahiro Nagahama, Keiko Kobayashi, Sadayuki Ochi and Masaya Takehara
Toxins 2021, 13(4), 272; https://doi.org/10.3390/toxins13040272 - 9 Apr 2021
Cited by 6 | Viewed by 3098
Abstract
Clostridium botulinum C2 toxin is a clostridial binary toxin consisting of actin ADP-ribosyltransferase (C2I) and C2II binding components. Activated C2II (C2IIa) binds to cellular receptors and forms oligomer in membrane rafts. C2IIa oligomer assembles with C2I and contributes to the transport of C2I [...] Read more.
Clostridium botulinum C2 toxin is a clostridial binary toxin consisting of actin ADP-ribosyltransferase (C2I) and C2II binding components. Activated C2II (C2IIa) binds to cellular receptors and forms oligomer in membrane rafts. C2IIa oligomer assembles with C2I and contributes to the transport of C2I into the cytoplasm of host cells. C2IIa induces Ca2+-induced lysosomal exocytosis, extracellular release of the acid sphingomyelinase (ASMase), and membrane invagination and endocytosis through generating ceramides in the membrane by ASMase. Here, we reveal that C2 toxin requires the lysosomal enzyme cathepsin B (CTSB) during endocytosis. Lysosomes are a rich source of proteases, containing cysteine protease CTSB and cathepsin L (CTSL), and aspartyl protease cathepsin D (CTSD). Cysteine protease inhibitor E64 blocked C2 toxin-induced cell rounding, but aspartyl protease inhibitor pepstatin-A did not. E64 inhibited the C2IIa-promoted extracellular ASMase activity, indicating that the protease contributes to the activation of ASMase. C2IIa induced the extracellular release of CTSB and CTSL, but not CTSD. CTSB knockdown by siRNA suppressed C2 toxin-caused cytotoxicity, but not siCTSL. These findings demonstrate that CTSB is important for effective cellular entry of C2 toxin into cells through increasing ASMase activity. Full article
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