163 results on '"Aggresomes"'
Search Results
2. The development of new biomarkers of spermatozoa quality in cattle
- Author
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Lindsey Fallon, Edgar Diaz-Miranda, Lauren Hamilton, Peter Sutovsky, Michal Zigo, Thomas E. Spencer, and M. Sofia Ortega
- Subjects
spermatozoa morphology ,sire fertility ,aggresomes ,semen processing ,flow cytometry ,Veterinary medicine ,SF600-1100 - Abstract
There is a current need for new biomarkers of spermatozoa quality, that consistently and correctly identify spermatozoa that will successfully contribute to subsequent embryo development. This could improve the standardization of semen analysis, decrease early embryo mortality, and use these biomarkers as a selection tool before servicing females. This study utilized imaging techniques to identify potential biomarkers of sperm quality, using sires previously classified as high (n = 4) or low (n = 4) performing at producing blastocysts in vitro. Spermatozoa were assessed before and following a gradient purification protocol, to understand how populations of cells are impacted by such protocols and may differ between in vivo and in vitro use. Pre-gradient samples from low-performing sires had an increased incidence of DNA damage, although post-gradient samples from high-performing sires were found to have an increased incidence of DNA damage. When evaluating morphology via fluorescent microscopy, the most prevalent defects in pre-gradient samples from high-performing sires were tail defects, which are successfully removed during purification processing. The most prevalent defects in pre-gradient samples from low-performing sires were aggresome defects located in the head, which would be brought into an oocyte upon fertilization and may impair embryo development. Image-based flow cytometry (IBFC) was employed to quantify defect prevalence to evaluate a greater sample size decreasing the variability that exists in manual assessments. Using IBFC, aggresome defects were again identified in the heads of spermatozoa from low-performing sires. Post-gradient samples from low-performing sires had a significantly greater (p
- Published
- 2023
- Full Text
- View/download PDF
3. β-Lapachone Exerts Anticancer Effects by Downregulating p53, Lys-Acetylated Proteins, TrkA, p38 MAPK, SOD1, Caspase-2, CD44 and NPM in Oxaliplatin-Resistant HCT116 Colorectal Cancer Cells.
- Author
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Jung, Eun Joo, Kim, Hye Jung, Shin, Sung Chul, Kim, Gon Sup, Jung, Jin-Myung, Hong, Soon Chan, Kim, Choong Won, and Lee, Won Sup
- Subjects
- *
CASPASES , *COLORECTAL cancer , *ANTINEOPLASTIC agents , *CD44 antigen , *CANCER cells , *IRINOTECAN , *CADHERINS - Abstract
β-lapachone (β-Lap), a topoisomerase inhibitor, is a naturally occurring ortho-naphthoquinone phytochemical and is involved in drug resistance mechanisms. Oxaliplatin (OxPt) is a commonly used chemotherapeutic drug for metastatic colorectal cancer, and OxPt-induced drug resistance remains to be solved to increase chances of successful therapy. To reveal the novel role of β-Lap associated with OxPt resistance, 5 μM OxPt-resistant HCT116 cells (HCT116-OxPt-R) were generated and characterized via hematoxylin staining, a CCK-8 assay and Western blot analysis. HCT116-OxPt-R cells were shown to have OxPt-specific resistance, increased aggresomes, upregulated p53 and downregulated caspase-9 and XIAP. Through signaling explorer antibody array, nucleophosmin (NPM), CD37, Nkx-2.5, SOD1, H2B, calreticulin, p38 MAPK, caspase-2, cadherin-9, MMP23B, ACOT2, Lys-acetylated proteins, COL3A1, TrkA, MPS-1, CD44, ITGA5, claudin-3, parkin and ACTG2 were identified as OxPt-R-related proteins due to a more than two-fold alteration in protein status. Gene ontology analysis suggested that TrkA, Nkx-2.5 and SOD1 were related to certain aggresomes produced in HCT116-OxPt-R cells. Moreover, β-Lap exerted more cytotoxicity and morphological changes in HCT116-OxPt-R cells than in HCT116 cells through the downregulation of p53, Lys-acetylated proteins, TrkA, p38 MAPK, SOD1, caspase-2, CD44 and NPM. Our results indicate that β-Lap could be used as an alternative drug to overcome the upregulated p53-containing OxPt-R caused by various OxPt-containing chemotherapies. [ABSTRACT FROM AUTHOR]
- Published
- 2023
- Full Text
- View/download PDF
4. Characterization of Skeletal Muscle Biopsy and Derived Myoblasts in a Patient Carrying Arg14del Mutation in Phospholamban Gene.
- Author
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Zanotti, Simona, Ripolone, Michela, Napoli, Laura, Velardo, Daniele, Salani, Sabrina, Ciscato, Patrizia, Priori, Silvia, Kukavica, Deni, Mazzanti, Andrea, Diamanti, Luca, Vegezzi, Elisa, Moggio, Maurizio, Corti, Stefania, Comi, Giacomo, and Sciacco, Monica
- Subjects
- *
SKELETAL muscle , *MYOBLASTS , *MYOCARDIUM , *GENETIC mutation , *DILATED cardiomyopathy , *NEMALINE myopathy - Abstract
Phospholamban is involved in the regulation of the activity and storage of calcium in cardiac muscle. Several mutations have been identified in the PLN gene causing cardiac disease associated with arrhythmogenic and dilated cardiomyopathy. The patho-mechanism underlying PLN mutations is not fully understood and a specific therapy is not yet available. PLN mutated patients have been deeply investigated in cardiac muscle, but very little is known about the effect of PLN mutations in skeletal muscle. In this study, we investigated both histological and functional features in skeletal muscle tissue and muscle-derived myoblasts from an Italian patient carrying the Arg14del mutation in PLN. The patient has a cardiac phenotype, but he also reported lower limb fatigability, cramps and fasciculations. The evaluation of a skeletal muscle biopsy showed histological, immunohistochemical and ultrastructural alterations. In particular, we detected an increase in the number of centronucleated fibers and a reduction in the fiber cross sectional area, an alteration in p62, LC3 and VCP proteins and the formation of perinuclear aggresomes. Furthermore, the patient's myoblasts showed a greater propensity to form aggresomes, even more marked after proteasome inhibition compared with control cells. Further genetic and functional studies are necessary to understand whether a definition of PLN myopathy, or cardiomyopathy plus, can be introduced for selected cases with clinical evidence of skeletal muscle involvement. Including skeletal muscle examination in the diagnostic process of PLN-mutated patients can help clarify this issue. [ABSTRACT FROM AUTHOR]
- Published
- 2023
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- View/download PDF
5. c-MYC-mediated TRIB3/P62+ aggresomes accumulation triggers paraptosis upon the combination of everolimus and ginsenoside Rh2
- Author
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Min-Xia Su, Yu-Lian Xu, Xiao-Ming Jiang, Mu-Yang Huang, Le-Le Zhang, Luo-Wei Yuan, Xiao-Huang Xu, Qi Zhu, Jian-Li Gao, Jia-Hong Lu, Xiuping Chen, Ming-Qing Huang, Yitao Wang, and Jin-Jian Lu
- Subjects
Everolimus ,Ginsenoside Rh2 ,Paraptosis ,Aggresomes ,P62 ,TRIB3 ,Therapeutics. Pharmacology ,RM1-950 - Abstract
The mammalian target of rapamycin (mTOR) pathway is abnormally activated in lung cancer. However, the anti-lung cancer effect of mTOR inhibitors as monotherapy is modest. Here, we identified that ginsenoside Rh2, an active component of Panax ginseng C. A. Mey., enhanced the anti-cancer effect of the mTOR inhibitor everolimus both in vitro and in vivo. Moreover, ginsenoside Rh2 alleviated the hepatic fat accumulation caused by everolimus in xenograft nude mice models. The combination of everolimus and ginsenoside Rh2 (labeled Eve-Rh2) induced caspase-independent cell death and cytoplasmic vacuolation in lung cancer cells, indicating that Eve-Rh2 prevented tumor progression by triggering paraptosis. Eve-Rh2 up-regulated the expression of c-MYC in cancer cells as well as tumor tissues. The increased c-MYC mediated the accumulation of tribbles homolog 3 (TRIB3)/P62+ aggresomes and consequently triggered paraptosis, bypassing the classical c-MYC/MAX pathway. Our study offers a potential effective and safe strategy for the treatment of lung cancer. Moreover, we have identified a new mechanism of TRIB3/P62+ aggresomes-triggered paraptosis and revealed a unique function of c-MYC.
- Published
- 2022
- Full Text
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6. Intracellular tau fragment droplets serve as seeds for tau fibrils.
- Author
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Soeda, Yoshiyuki, Yoshimura, Hideaki, Bannai, Hiroko, Koike, Riki, Shiiba, Isshin, and Takashima, Akihiko
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BLUE light , *ALZHEIMER'S disease , *TAU proteins , *NEUROFIBRILLARY tangles , *PHASE separation , *NEURODEGENERATION - Abstract
Intracellular tau aggregation requires a local protein concentration increase, referred to as "droplets". However, the cellular mechanism for droplet formation is poorly understood. Here, we expressed OptoTau, a P301L mutant tau fused with CRY2olig, a light-sensitive protein that can form homo-oligomers. Under blue light exposure, OptoTau increased tau phosphorylation and was sequestered in aggresomes. Suppressing aggresome formation by nocodazole formed tau granular clusters in the cytoplasm. The granular clusters disappeared by discontinuing blue light exposure or 1,6-hexanediol treatment suggesting that intracellular tau droplet formation requires microtubule collapse. Expressing OptoTau-ΔN, a species of N-terminal cleaved tau observed in the Alzheimer's disease brain, formed 1,6-hexanediol and detergent-resistant tau clusters in the cytoplasm with blue light stimulation. These intracellular stable tau clusters acted as a seed for tau fibrils in vitro. These results suggest that tau droplet formation and N-terminal cleavage are necessary for neurofibrillary tangles formation in neurodegenerative diseases. [Display omitted] • OptoTau, P301Ltau fused with CRY2olig, was sequestered in aggresomes by blue light • OptoTau formed droplets upon exposure to blue light under disrupted aggresomes • N-terminal cleaved OptoTau (OptoTau-ΔN) formed detergent-insoluble aggregates • Intracellular OptoTau-ΔN aggregates acted as seeds for tau fibrils Soeda et al. propose a new model for cellular tau aggregation using OptoTau, where P301L tau is fused with CRY2olig, a light-sensitive optogenetics tool. According to this model, tau droplet formation through a disruption of aggresomes and N-terminal cleavage are necessary for the formation of neurofibrillary tangles in neurodegenerative diseases. [ABSTRACT FROM AUTHOR]
- Published
- 2024
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7. Extensive accumulation of misfolded protein aggregates during natural aging and senescence
- Author
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Karina Cuanalo-Contreras, Jonathan Schulz, Abhisek Mukherjee, Kyung-Won Park, Enrique Armijo, and Claudio Soto
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protein misfolding ,amyloid ,prions ,aggresomes ,senescence ,proteostasis ,Neurosciences. Biological psychiatry. Neuropsychiatry ,RC321-571 - Abstract
Accumulation of misfolded protein aggregates is a hallmark event in many age-related protein misfolding disorders, including some of the most prevalent and insidious neurodegenerative diseases. Misfolded protein aggregates produce progressive cell damage, organ dysfunction, and clinical changes, which are common also in natural aging. Thus, we hypothesized that aging is associated to the widespread and progressive misfolding and aggregation of many proteins in various tissues. In this study, we analyzed whether proteins misfold, aggregate, and accumulate during normal aging in three different biological systems, namely senescent cells, Caenorhabditis elegans, and mouse tissues collected at different times from youth to old age. Our results show a significant accumulation of misfolded protein aggregates in aged samples as compared to young materials. Indeed, aged samples have between 1.3 and 2.5-fold (depending on the biological system) higher amount of insoluble proteins than young samples. These insoluble proteins exhibit the typical characteristics of disease-associated aggregates, including insolubility in detergents, protease resistance, and staining with amyloid-binding dye as well as accumulation in aggresomes. We identified the main proteins accumulating in the aging brain using proteomic studies. These results show that the aged brain contain large amounts of misfolded and likely non-functional species of many proteins, whose soluble versions participate in cellular pathways that play fundamental roles in preserving basic functions, such as protein quality control, synapsis, and metabolism. Our findings reveal a putative role for protein misfolding and aggregation in aging.
- Published
- 2023
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8. β-Lapachone Exerts Anticancer Effects by Downregulating p53, Lys-Acetylated Proteins, TrkA, p38 MAPK, SOD1, Caspase-2, CD44 and NPM in Oxaliplatin-Resistant HCT116 Colorectal Cancer Cells
- Author
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Eun Joo Jung, Hye Jung Kim, Sung Chul Shin, Gon Sup Kim, Jin-Myung Jung, Soon Chan Hong, Choong Won Kim, and Won Sup Lee
- Subjects
β-lapachone ,anticancer effect ,oxaliplatin-resistant ,colorectal cancer ,p53 ,aggresomes ,Biology (General) ,QH301-705.5 ,Chemistry ,QD1-999 - Abstract
β-lapachone (β-Lap), a topoisomerase inhibitor, is a naturally occurring ortho-naphthoquinone phytochemical and is involved in drug resistance mechanisms. Oxaliplatin (OxPt) is a commonly used chemotherapeutic drug for metastatic colorectal cancer, and OxPt-induced drug resistance remains to be solved to increase chances of successful therapy. To reveal the novel role of β-Lap associated with OxPt resistance, 5 μM OxPt-resistant HCT116 cells (HCT116-OxPt-R) were generated and characterized via hematoxylin staining, a CCK-8 assay and Western blot analysis. HCT116-OxPt-R cells were shown to have OxPt-specific resistance, increased aggresomes, upregulated p53 and downregulated caspase-9 and XIAP. Through signaling explorer antibody array, nucleophosmin (NPM), CD37, Nkx-2.5, SOD1, H2B, calreticulin, p38 MAPK, caspase-2, cadherin-9, MMP23B, ACOT2, Lys-acetylated proteins, COL3A1, TrkA, MPS-1, CD44, ITGA5, claudin-3, parkin and ACTG2 were identified as OxPt-R-related proteins due to a more than two-fold alteration in protein status. Gene ontology analysis suggested that TrkA, Nkx-2.5 and SOD1 were related to certain aggresomes produced in HCT116-OxPt-R cells. Moreover, β-Lap exerted more cytotoxicity and morphological changes in HCT116-OxPt-R cells than in HCT116 cells through the downregulation of p53, Lys-acetylated proteins, TrkA, p38 MAPK, SOD1, caspase-2, CD44 and NPM. Our results indicate that β-Lap could be used as an alternative drug to overcome the upregulated p53-containing OxPt-R caused by various OxPt-containing chemotherapies.
- Published
- 2023
- Full Text
- View/download PDF
9. Characterization of Skeletal Muscle Biopsy and Derived Myoblasts in a Patient Carrying Arg14del Mutation in Phospholamban Gene
- Author
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Simona Zanotti, Michela Ripolone, Laura Napoli, Daniele Velardo, Sabrina Salani, Patrizia Ciscato, Silvia Priori, Deni Kukavica, Andrea Mazzanti, Luca Diamanti, Elisa Vegezzi, Maurizio Moggio, Stefania Corti, Giacomo Comi, and Monica Sciacco
- Subjects
phospholamban ,Arg14del ,skeletal muscle ,aggresomes ,Cytology ,QH573-671 - Abstract
Phospholamban is involved in the regulation of the activity and storage of calcium in cardiac muscle. Several mutations have been identified in the PLN gene causing cardiac disease associated with arrhythmogenic and dilated cardiomyopathy. The patho-mechanism underlying PLN mutations is not fully understood and a specific therapy is not yet available. PLN mutated patients have been deeply investigated in cardiac muscle, but very little is known about the effect of PLN mutations in skeletal muscle. In this study, we investigated both histological and functional features in skeletal muscle tissue and muscle-derived myoblasts from an Italian patient carrying the Arg14del mutation in PLN. The patient has a cardiac phenotype, but he also reported lower limb fatigability, cramps and fasciculations. The evaluation of a skeletal muscle biopsy showed histological, immunohistochemical and ultrastructural alterations. In particular, we detected an increase in the number of centronucleated fibers and a reduction in the fiber cross sectional area, an alteration in p62, LC3 and VCP proteins and the formation of perinuclear aggresomes. Furthermore, the patient’s myoblasts showed a greater propensity to form aggresomes, even more marked after proteasome inhibition compared with control cells. Further genetic and functional studies are necessary to understand whether a definition of PLN myopathy, or cardiomyopathy plus, can be introduced for selected cases with clinical evidence of skeletal muscle involvement. Including skeletal muscle examination in the diagnostic process of PLN-mutated patients can help clarify this issue.
- Published
- 2023
- Full Text
- View/download PDF
10. c-MYC-mediated TRIB3/P62+ aggresomes accumulation triggers paraptosis upon the combination of everolimus and ginsenoside Rh2.
- Author
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Su, Min-Xia, Xu, Yu-Lian, Jiang, Xiao-Ming, Huang, Mu-Yang, Zhang, Le-Le, Yuan, Luo-Wei, Xu, Xiao-Huang, Zhu, Qi, Gao, Jian-Li, Lu, Jia-Hong, Chen, Xiuping, Huang, Ming-Qing, Wang, Yitao, and Lu, Jin-Jian
- Subjects
GINSENOSIDES ,EVEROLIMUS ,ANTINEOPLASTIC agents ,GINSENG ,MTOR inhibitors ,LUNG cancer - Abstract
The mammalian target of rapamycin (mTOR) pathway is abnormally activated in lung cancer. However, the anti-lung cancer effect of mTOR inhibitors as monotherapy is modest. Here, we identified that ginsenoside Rh2, an active component of Panax ginseng C. A. Mey., enhanced the anti-cancer effect of the mTOR inhibitor everolimus both in vitro and in vivo. Moreover, ginsenoside Rh2 alleviated the hepatic fat accumulation caused by everolimus in xenograft nude mice models. The combination of everolimus and ginsenoside Rh2 (labeled Eve-Rh2) induced caspase-independent cell death and cytoplasmic vacuolation in lung cancer cells, indicating that Eve-Rh2 prevented tumor progression by triggering paraptosis. Eve-Rh2 up-regulated the expression of c-MYC in cancer cells as well as tumor tissues. The increased c-MYC mediated the accumulation of tribbles homolog 3 (TRIB3)/P62
+ aggresomes and consequently triggered paraptosis, bypassing the classical c-MYC/MAX pathway. Our study offers a potential effective and safe strategy for the treatment of lung cancer. Moreover, we have identified a new mechanism of TRIB3/P62+ aggresomes-triggered paraptosis and revealed a unique function of c-MYC. Eve-Rh2 up-regulates the expression of c-MYC, which mediates the accumulation of TRIB3/P62+ aggresomes bypassing the classical c-MYC/MAX pathway, thereby triggering paraptosis and ultimately inhibiting tumor progression. [Display omitted] [ABSTRACT FROM AUTHOR]- Published
- 2022
- Full Text
- View/download PDF
11. The E3 Ubiquitin Ligase TRAF6 Interacts with the Cellular Prion Protein and Modulates Its Solubility and Recruitment to Cytoplasmic p62/SQSTM1-Positive Aggresome-Like Structures.
- Author
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Masperone, Lara, Codrich, Marta, Persichetti, Francesca, Gustincich, Stefano, Zucchelli, Silvia, and Legname, Giuseppe
- Abstract
The cellular prion protein (PrP
C ) is a ubiquitous glycoprotein highly expressed in the brain where it is involved in neurite outgrowth, copper homeostasis, NMDA receptor regulation, cell adhesion, and cell signaling. Conformational conversion of PrPC into its insoluble and aggregation-prone scrapie form (PrPSc ) is the trigger for several rare devastating neurodegenerative disorders, collectively referred to as prion diseases. Recent work indicates that the ubiquitin–proteasome system is involved in quality control of PrPC . To better dissect the role of ubiquitination in PrPC physiology, we focused on the E3 RING ubiquitin ligase tumor necrosis factor receptor (TNFR)-associated factor 6 (TRAF6). Here, we report that PrPC interacts with TRAF6 both in vitro, in cells, and in vivo, in the mouse brain. Transient overexpression of TRAF6 indirectly modulates PrPC ubiquitination and triggers redistribution of PrPC into the insoluble fraction. Importantly, in the presence of wild-type TRAF6, but not a mutant lacking E3 ligase activity, PrPC accumulates into cytoplasmic aggresome-like inclusions containing TRAF6 and p62/SQSTM1. Our results suggest that TRAF6 ligase activity could exert a role in the regulation of PrPC redistribution in cells under physiological conditions. This novel interaction may uncover possible mechanisms of cell clearance/reorganization in prion diseases. [ABSTRACT FROM AUTHOR]- Published
- 2022
- Full Text
- View/download PDF
12. Defective transcription elongation in human cancers imposes targetable proteotoxic vulnerability
- Author
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B. Muhammad, L.G. Parks, K. Komurov, and L.M. Privette Vinnedge
- Subjects
Transcription elongation defects (TEdef) ,Aggresomes ,Protein homeostasis ,Proteotoxic stress ,Autophagy ,Neoplasms. Tumors. Oncology. Including cancer and carcinogens ,RC254-282 - Abstract
Successful cancer therapy is contingent on identifying cancer-specific aberrant phenotypes and their associated vulnerabilities. We recently reported that a subset of almost every cancer type contains a genome-wide defect in RNA Polymerase II-mediated transcription elongation (TEdef), which impairs the expression of long genes and confers resistance to anti-tumor immune attack. Using a combination of computational analysis and laboratory experiments, we report that tumor cells with TEdef have widespread overexpression of the components of the protein homeostasis machinery (mostly composed of short genes), including protein folding and clearance. Accordingly, TEdef cells were characterized by abnormally high levels of insoluble protein aggregates in the cytoplasm and autophagy influx. We present evidence that TEdef cells exhibit impaired clearance of misfolded protein aggregates through the ubiquitin-proteasome system, and thus rely on autophagy for their degradation. As such, while these cells were highly resistant to proteasome inhibitors, they were acutely sensitive to inhibitors of autophagy in vitro and in vivo. This study reveals a major aberrant phenotype that is observed in ∼15–25% of all cancers and characterizes a unique cellular vulnerability that can be readily exploited in the clinic to improve treatment efficacy.
- Published
- 2022
- Full Text
- View/download PDF
13. Cytosolic Nipah Virus Inclusion Bodies Recruit Proteins Without Using Canonical Aggresome Pathways
- Author
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Nico Becker, Anja Heiner, and Andrea Maisner
- Subjects
Nipah virus ,inclusions ,aggresomes ,HDAC6 ,BAG3 ,microtubules ,Microbiology ,QR1-502 - Abstract
Nipah virus (NiV) is a BSL-4 classified zoonotic paramyxovirus that causes respiratory or encephalitic diseases. A hallmark of NiV infections, as with all cell infections caused by non-segmented negative-strand RNA viruses, is the formation of cytoplasmic inclusion bodies (IBs). We previously showed that cytosolic NiV IBs, which are formed in infected cells or in cells minimally expressing the NiV nucleocapsid proteins, are associated with the microtubule-organizing center (MTOC) marker γ-tubulin. They also recruit overexpressed cytosolic proteins that are not functionally required for viral replication in IBs and that otherwise might form toxic protein aggregates. Therefore, NiV IBs are thought to share some functional properties with cellular aggresomes. The fact that aggresomes were not found in NiV-infected cells supports the idea that NiV IBs are successfully reducing the proteotoxic stress in infected cells. Only if the proteasome-ubiquitin system is artificially blocked by inhibitors, cellular aggresomes are formed in addition to IBs, but without colocalizing. Although both structures were positive for the classical aggresome markers histone deacetylase 6 (HDAC6) and Bcl-2-associated athanogene 3 (BAG3), they clearly differed in their cellular protein compositions and recruited overexpressed proteins to different extents. The further finding that inhibition of aggresome pathways by HDAC6 or microtubule (MT) inhibitors did neither interfere with IB formation nor with protein sequestration, strengthens the idea that cytosolic NiV IBs can assume some aggresome-like functions without involving active transport processes and canonical cellular aggresome pathways.
- Published
- 2022
- Full Text
- View/download PDF
14. Trafficking of Full-Length and N-Terminally Truncated Cathepsin B in Human Colorectal Carcinoma Cells.
- Author
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Tamhane, Tripti, Njenga, Robin W., Burden, Roberta E., Büth, Heiko, Maelandsmo, Gunhild M., Haugen, Mads H., Scott, Christopher J., and Brix, Klaudia
- Subjects
CATHEPSIN B ,COLORECTAL cancer ,PEPTIDES ,GREEN fluorescent protein ,CELLULAR inclusions - Abstract
Featured Application: Full-length cathepsin B is sorted to endo-lysosomes of colorectal carcinoma cells, while N-terminally truncated cathepsin B accumulates in aggresomes. Cathepsin B is an endo-lysosomal cysteine protease. However, its increased expression and altered localization to the extracellular space, to mitochondria, or to the nucleus has been linked to tumor progression. In the present study, we show enhanced levels of cathepsin B in adenocarcinoma tissue in comparison to adjacent normal colon. Additionally, cathepsin B was observed in the nuclear compartment of mucosal cells in adenocarcinoma tissue samples and in the nuclei of the colorectal carcinoma cell line HCT116. Accordingly, a distinct 40-kDa form of cathepsin B was detected in HCT116 cells, which is proposed to represent a specific form lacking the signal peptide and parts of the propeptide. Trafficking studies with an EGFP-tagged N-terminally truncated form, mimicking the 40-kDa form, demonstrated accumulation in aggresome-like inclusion bodies, while EGFP-tagged full-length cathepsin B revealed regular sorting to endo-lysosomes. We conclude that the identity of nuclear cathepsin B in colorectal adenocarcinoma (in situ) and in carcinoma cells (in vitro) cannot be attributed to either full-length or 40-kDa N-terminally truncated cathepsin B forms. Hence, future studies are needed to demonstrate which form/s of cathepsin B may be sorted to the nuclei of colorectal carcinoma cells, and whether redundant regulation of related cathepsin expression occurs. [ABSTRACT FROM AUTHOR]
- Published
- 2021
- Full Text
- View/download PDF
15. LC3A-mediated autophagy elicits PERK-eIF2α-ATF4 axis activation and mitochondrial dysfunction: Exposing vulnerability in aggresome-positive cancer cells.
- Author
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Amer N, Hesham D, Al-Shehaby N, Elshoky HA, Amer M, Magdeldin S, Mansour M, Abou-Aisha K, and El-Naggar S
- Subjects
- Humans, Cell Line, Tumor, Unfolded Protein Response, Neoplasms metabolism, Neoplasms pathology, Neoplasms genetics, Autophagy, Activating Transcription Factor 4 metabolism, Activating Transcription Factor 4 genetics, eIF-2 Kinase metabolism, eIF-2 Kinase genetics, Mitochondria metabolism, Mitochondria pathology, Microtubule-Associated Proteins metabolism, Microtubule-Associated Proteins genetics, Eukaryotic Initiation Factor-2 metabolism, Eukaryotic Initiation Factor-2 genetics
- Abstract
The unfolded protein response pathways (UPR), autophagy, and compartmentalization of misfolded proteins into inclusion bodies are critical components of the protein quality control network. Among inclusion bodies, aggresomes are particularly intriguing due to their association with cellular survival, drug resistance, and aggresive cancer behavior. Aggresomes are molecular condensates formed when collapsed vimentin cages encircle misfolded proteins before final removal by autophagy. Yet significant gaps persist in the mechanisms governing aggresome formation and elimination in cancer cells. Understanding these mechanisms is crucial, especially considering the involvement of LC3A, a member of the MAP1LC3 family, which plays a unique role in autophagy regulation and has been reported to be epigenetically silenced in many cancers. Herein, we utilized the tetracycline-inducible expression of LC3A to investigate its role in choroid plexus carcinoma cells, which inherently exhibit the presence of aggresomes. Live cell imaging was employed to demonstrate the effect of LC3A expression on aggresome-positive cells, while SILAC-based proteomics identified LC3A-induced protein and pathway alterations. Our findings demonstrated that extended expression of LC3A is associated with cellular senescence. However, the obstruction of lysosomal degradation in this context has a deleterious effect on cellular viability. In response to LC3A-induced autophagy, we observed significant alterations in mitochondrial morphology, reflected by mitochondrial dysfunction and increased ROS production. Furthermore, LC3A expression elicited the activation of the PERK-eIF2α-ATF4 axis of the UPR, underscoring a significant change in the protein quality control network. In conclusion, our results elucidate that LC3A-mediated autophagy alters the protein quality control network, exposing a vulnerability in aggresome-positive cancer cells., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)
- Published
- 2024
- Full Text
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16. Aggresomes predict poor outcomes and implicate proteostasis in the pathogenesis of pediatric choroid plexus tumors.
- Author
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Amer, Nada, Taha, Hala, Hesham, Dina, Al-Shehaby, Nouran, Mosaab, Amal, Soudy, Mohamed, Osama, Aya, Mahmoud, Noura, Elayadi, Moatasem, Youssef, Ayda, Elbeltagy, Mohamed, Zaghloul, Mohamed Saad, Magdeldin, Sameh, Sayed, Ahmed A., and El-Naggar, Shahenda
- Abstract
Purpose: Protein misfolding and aggregation result in proteotoxic stress and underlie the pathogenesis of many diseases. To overcome proteotoxicity, cells compartmentalize misfolded and aggregated proteins in different inclusion bodies. The aggresome is a paranuclear inclusion body that functions as a storage compartment for misfolded proteins. Choroid plexus tumors (CPTs) are rare neoplasms comprised of three pathological subgroups. The underlying mechanisms of their pathogenesis remain unclear. This study aims to elucidate the prognostic role and the biological effects of aggresomes in pediatric CPTs. Methods: We examined the presence of aggresomes in 42 patient-derived tumor tissues by immunohistochemistry and we identified their impact on patients' outcomes. We then investigated the proteogenomics signature associated with aggresomes using whole-genome DNA methylation and proteomic analysis to define their role in the pathogenesis of pediatric CPTs. Results: Aggresomes were detected in 64.2% of samples and were distributed among different pathological and molecular subgroups. The presence of aggresomes with different percentages was correlated with patients' outcomes. The ≥ 25% cutoff had the most significant impact on overall and event-free survival (p-value < 0.001) compared to the pathological and the molecular stratifications. Conclusions: These results support the role of aggresome as a novel prognostic molecular marker for pediatric CPTs that was comparable to the molecular classification in segregating samples into two distinct subgroups, and to the pathological stratification in the prediction of patients' outcomes. Moreover, the proteogenomic signature of CPTs displayed altered protein homeostasis, manifested by enrichment in processes related to protein quality control. [ABSTRACT FROM AUTHOR]
- Published
- 2021
- Full Text
- View/download PDF
17. Trafficking of Full-Length and N-Terminally Truncated Cathepsin B in Human Colorectal Carcinoma Cells
- Author
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Tripti Tamhane, Robin W. Njenga, Roberta E. Burden, Heiko Büth, Gunhild M. Maelandsmo, Mads H. Haugen, Christopher J. Scott, and Klaudia Brix
- Subjects
aggresomes ,cathepsin B ,colorectal cancer ,enhanced green fluorescent protein tagging ,nuclear specific protease forms ,protein engineering ,Technology ,Engineering (General). Civil engineering (General) ,TA1-2040 ,Biology (General) ,QH301-705.5 ,Physics ,QC1-999 ,Chemistry ,QD1-999 - Abstract
Cathepsin B is an endo-lysosomal cysteine protease. However, its increased expression and altered localization to the extracellular space, to mitochondria, or to the nucleus has been linked to tumor progression. In the present study, we show enhanced levels of cathepsin B in adenocarcinoma tissue in comparison to adjacent normal colon. Additionally, cathepsin B was observed in the nuclear compartment of mucosal cells in adenocarcinoma tissue samples and in the nuclei of the colorectal carcinoma cell line HCT116. Accordingly, a distinct 40-kDa form of cathepsin B was detected in HCT116 cells, which is proposed to represent a specific form lacking the signal peptide and parts of the propeptide. Trafficking studies with an EGFP-tagged N-terminally truncated form, mimicking the 40-kDa form, demonstrated accumulation in aggresome-like inclusion bodies, while EGFP-tagged full-length cathepsin B revealed regular sorting to endo-lysosomes. We conclude that the identity of nuclear cathepsin B in colorectal adenocarcinoma (in situ) and in carcinoma cells (in vitro) cannot be attributed to either full-length or 40-kDa N-terminally truncated cathepsin B forms. Hence, future studies are needed to demonstrate which form/s of cathepsin B may be sorted to the nuclei of colorectal carcinoma cells, and whether redundant regulation of related cathepsin expression occurs.
- Published
- 2021
- Full Text
- View/download PDF
18. Cytokinin‐induced protein synthesis suppresses growth and osmotic stress tolerance.
- Author
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Karunadasa, Sumudu S., Kurepa, Jasmina, Shull, Timothy E., and Smalle, Jan A.
- Subjects
- *
ABSCISIC acid , *CYTOKININS , *PROTEIN synthesis , *OSMOTIC pressure , *OSMOREGULATION , *SENSITIVE plant , *RIBOSOMAL proteins , *PLANT development , *PLANT growth - Abstract
Summary: Cytokinins control critical aspects of plant development and environmental responses. Perception of cytokinin ultimately leads to the activation of proteins belonging to the type‐B Response Regulator family of cytokinin response activators. In Arabidopsis thaliana, ARR1 is one of the most abundantly expressed type‐B Response Regulators.We investigated the link between cytokinin signaling, protein synthesis, plant growth and osmotic stress tolerance.We show that the increased cytokinin signaling in ARR1 gain‐of‐function transgenic lines is associated with increased rates of protein synthesis, which lead to growth inhibition and hypersensitivity to osmotic stress. Cytokinin‐induced growth inhibition and osmotic stress hypersensitivity were rescued by treatments with ABA, a hormone known to inhibit protein synthesis. We also demonstrate that cytokinin‐induced protein synthesis requires isoforms of the ribosomal protein L4 encoded by the cytokinin‐inducible genes RPL4A and RPL4D, and that RPL4 loss‐of‐function increases osmotic stress tolerance and decreases sensitivity to cytokinin‐induced growth inhibition.These findings reveal that an increase in protein synthesis negatively impacts growth and osmotic stress tolerance and explain some of the adverse effects of elevated cytokinin action on plant development and stress physiology. [ABSTRACT FROM AUTHOR]
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- 2020
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19. β-Lapachone Exerts Anticancer Effects by Downregulating p53, Lys-Acetylated Proteins, TrkA, p38 MAPK, SOD1, Caspase-2, CD44 and NPM in Oxaliplatin-Resistant HCT116 Colorectal Cancer Cells
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Lee, Eun Joo Jung, Hye Jung Kim, Sung Chul Shin, Gon Sup Kim, Jin-Myung Jung, Soon Chan Hong, Choong Won Kim, and Won Sup
- Subjects
β-lapachone ,anticancer effect ,oxaliplatin-resistant ,colorectal cancer ,p53 ,aggresomes ,antibody array ,chemotherapy - Abstract
β-lapachone (β-Lap), a topoisomerase inhibitor, is a naturally occurring ortho-naphthoquinone phytochemical and is involved in drug resistance mechanisms. Oxaliplatin (OxPt) is a commonly used chemotherapeutic drug for metastatic colorectal cancer, and OxPt-induced drug resistance remains to be solved to increase chances of successful therapy. To reveal the novel role of β-Lap associated with OxPt resistance, 5 μM OxPt-resistant HCT116 cells (HCT116-OxPt-R) were generated and characterized via hematoxylin staining, a CCK-8 assay and Western blot analysis. HCT116-OxPt-R cells were shown to have OxPt-specific resistance, increased aggresomes, upregulated p53 and downregulated caspase-9 and XIAP. Through signaling explorer antibody array, nucleophosmin (NPM), CD37, Nkx-2.5, SOD1, H2B, calreticulin, p38 MAPK, caspase-2, cadherin-9, MMP23B, ACOT2, Lys-acetylated proteins, COL3A1, TrkA, MPS-1, CD44, ITGA5, claudin-3, parkin and ACTG2 were identified as OxPt-R-related proteins due to a more than two-fold alteration in protein status. Gene ontology analysis suggested that TrkA, Nkx-2.5 and SOD1 were related to certain aggresomes produced in HCT116-OxPt-R cells. Moreover, β-Lap exerted more cytotoxicity and morphological changes in HCT116-OxPt-R cells than in HCT116 cells through the downregulation of p53, Lys-acetylated proteins, TrkA, p38 MAPK, SOD1, caspase-2, CD44 and NPM. Our results indicate that β-Lap could be used as an alternative drug to overcome the upregulated p53-containing OxPt-R caused by various OxPt-containing chemotherapies.
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- 2023
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20. Upregulation of brain cholesterol levels inhibits mitophagy in Alzheimer disease.
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Roca-Agujetas, Vicente, de Dios, Cristina, Abadin, Xenia, and Colell, Anna
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MITOCHONDRIAL pathology ,ALZHEIMER'S disease ,CHOLESTEROL ,CYTOSOL ,GLUTATHIONE - Abstract
Mitochondrial dysfunction is behind several neurodegenerative diseases, including Alzheimer disease (AD). Accumulation of damaged mitochondria is already observed at the early stages of AD and has been linked to impaired mitophagy, but the mechanisms underlying this alteration are still not fully known. In our recent study, we show that intracellular cholesterol enrichment can downregulate amyloid beta (Aβ)-induced mitophagy. Mitochondrial glutathione depletion resulting from high cholesterol levels promotes PINK1 (PTEN induced kinase 1)-mediated mitophagosome formation; however, mitophagy flux is ultimately disrupted, most likely due to fusion deficiency of endosomes-lysosomes caused by cholesterol. Meanwhile, in APP-PSEN1-SREBF2 mice, an AD mouse model that overexpresses the cholesterol-related transcription factor SREBF2, cholesterol accumulation prompts an oxidative- and age-dependent cytosolic aggregation of the mitophagy adaptor OPTN (optineurin), which prevents mitophagosome formation despite enhanced PINK1-PRKN/parkin signaling. Hippocampal neurons from postmortem brain of AD individuals reproduce the progressive accumulation of OPTN in aggresome-like structures accompanied by high levels of mitochondrial cholesterol in advanced stages of the disease. Overall, these data provide new insights into the impairment of the PINK1-PRKN mitophagy pathway in AD and suggest the combination of mitophagy inducers with strategies focused on restoring the cholesterol homeostasis and mitochondrial redox balance as a potential disease-modifying therapy for AD. [ABSTRACT FROM AUTHOR]
- Published
- 2021
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21. USP1 (ubiquitin specific peptidase 1) targets ULK1 and regulates its cellular compartmentalization and autophagy.
- Author
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Raimondi, Marzia, Cesselli, Daniela, Di Loreto, Carla, La Marra, Francesco, Schneider, Claudio, and Demarchi, Francesca
- Abstract
ULK1 (unc-51 like autophagy activating kinase 1) is a core component at multiple steps of canonical macroautophagy/autophagy. The activity of ULK1 is tightly regulated by several post-translational modifications, including ubiquitination, yet the deubiquitinase (DUB) responsible for its reversible deubiquitination has not been described. Here, we identified USP1 (ubiquitin specific peptidase 1) as a key player in the modulation of ULK1 K63-linked deubiquitination. Moreover, both USP1 depletion and its chemical inhibition by pimozide are coupled to a reduction of ULK1 in Triton X-100 soluble cellular lysates, and its compartmentalization to a fraction that can be solubilized in 5 M urea. In USP1-depleted cells this fraction is also enriched in SQSTM1 (sequestosome 1), the aggresome marker HDAC6 (histone deacetylase 6), and the prototype of USP1 targets FANCD2 (FA complementation group D2). Consistently, in USP1-depleted and pimozide-treated cells, ULK1 forms protein aggregates enriched in SQSTM1, as detected by both immummunofluorescence and co-immunoprecipitation studies. Notably, depletion of USP1 inhibits canonical autophagic flux and promotes an alternative route leading to lysosomal-mediated degradation of SQSTM1. Our findings reveal a novel function of the USP1-ULK1 axis as a modulator of the switch between canonical and unconventional autophagy. Further, we provide the first evidence supporting the existence of a subset of breast tumors co-expressing ULK1 and MAP1LC3B (microtubule associated protein 1 light chain 3 beta) proteins. Because the USP1 inhibitor pimozide affects breast cancer cell growth, targeting USP1 in those tumors relying on autophagy for growth might prove to be a convenient therapeutic strategy. Abbreviations: ATG13: autophagy related 13; BECN1: beclin 1; BZ: bortezomib; CAPN1: calpain 1; DUB: deubiquitinase; FANCI: FA complementation group I; FANCD2: FA complementation group D2; FZR1: fizzy and cell division cycle 20 related 1; HDAC6: histone deacetylase 6; MAP1LC3B: microtubule associated protein 1 light chain 3 beta; PMZ: pimozide; SH3GLB1: SH3 domain containing GRB2 like, endophilin B1; SQSTM1: sequestosome 1; TRAF6: TNF receptor associated factor 6; ULK1: unc-51 like autophagy activating kinase 1; USP1: ubiquitin specific peptidase 1; WDR48: WD repeat domain 48 [ABSTRACT FROM AUTHOR]
- Published
- 2019
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22. The Nucleolus as a Stress Response Organelle
- Author
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Lindström, Mikael S., Latonen, Leena, O'Day, Danton H, editor, and Catalano, Andrew, editor
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- 2013
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23. The development of new biomarkers of spermatozoa quality in cattle.
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Fallon L, Diaz-Miranda E, Hamilton L, Sutovsky P, Zigo M, Spencer TE, and Ortega MS
- Abstract
There is a current need for new biomarkers of spermatozoa quality, that consistently and correctly identify spermatozoa that will successfully contribute to subsequent embryo development. This could improve the standardization of semen analysis, decrease early embryo mortality, and use these biomarkers as a selection tool before servicing females. This study utilized imaging techniques to identify potential biomarkers of sperm quality, using sires previously classified as high ( n = 4) or low ( n = 4) performing at producing blastocysts in vitro. Spermatozoa were assessed before and following a gradient purification protocol, to understand how populations of cells are impacted by such protocols and may differ between in vivo and in vitro use. Pre-gradient samples from low-performing sires had an increased incidence of DNA damage, although post-gradient samples from high-performing sires were found to have an increased incidence of DNA damage. When evaluating morphology via fluorescent microscopy, the most prevalent defects in pre-gradient samples from high-performing sires were tail defects, which are successfully removed during purification processing. The most prevalent defects in pre-gradient samples from low-performing sires were aggresome defects located in the head, which would be brought into an oocyte upon fertilization and may impair embryo development. Image-based flow cytometry (IBFC) was employed to quantify defect prevalence to evaluate a greater sample size decreasing the variability that exists in manual assessments. Using IBFC, aggresome defects were again identified in the heads of spermatozoa from low-performing sires. Post-gradient samples from low-performing sires had a significantly greater ( p < 0.05) incidence of aggresome defects than post-gradient samples from high-performing sires. Additionally, IBFC was used to evaluate spermatozoa viability following gradient purification. Distinct populations of sperm cells were identified. High-performing sires had more spermatozoa in the population deemed most viable than low-performing sires. This study demonstrated that spermatozoa defects vary in populations before and following gradient purification, indicating that it may be beneficial to separately evaluate semen for in vivo and in vitro use. Furthermore, a prevalent defect in low-performing sires that could explain a discrepancy between successful fertilization and embryo development was identified. Therefore, elucidating a malfunction regulated by sire, that could potentially affect early embryo development., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Fallon, Diaz-Miranda, Hamilton, Sutovsky, Zigo, Spencer and Ortega.)
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- 2023
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24. A heterozygous p.S143P mutation in LMNA associates with proteasome dysfunction and enhanced autophagy-mediated degradation of mutant lamins A and C
- Author
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West, Gun, Turunen, Minttu, Aalto, Anna, Virtanen, Laura, Li, Song-Ping, Heliö, Tiina, Meinander, Annika, Taimen, Pekka, HUS Heart and Lung Center, Clinicum, Department of Medicine, and Helsinki University Hospital Area
- Subjects
autophagy ,CARDIOMYOPATHY ,STRESS ,ubiquitin (Ub) ,UBIQUITIN ,DISEASE ,ubiquitin-proteasome degradation system ,FUSION ,QUALITY CONTROL ,AGGRESOMES ,lamin A/C (LMNA) ,1182 Biochemistry, cell and molecular biology ,disease mutations ,INHIBITORS ,SYSTEM ,degradation ,ACCUMULATION - Abstract
Lamins A and C are nuclear intermediate filament proteins that form a proteinaceous meshwork called lamina beneath the inner nuclear membrane. Mutations in the LMNA gene encoding lamins A and C cause a heterogenous group of inherited degenerative diseases known as laminopathies. Previous studies have revealed altered cell signaling pathways in lamin-mutant patient cells, but little is known about the fate of mutant lamins A and C within the cells. Here, we analyzed the turnover of lamins A and C in cells derived from a dilated cardiomyopathy patient with a heterozygous p.S143P mutation in LMNA. We found that transcriptional activation and mRNA levels of LMNA are increased in the primary patient fibroblasts, but the protein levels of lamins A and C remain equal in control and patient cells because of a meticulous interplay between autophagy and the ubiquitin-proteasome system (UPS). Both endogenous and ectopic expression of p.S143P lamins A and C cause significantly reduced activity of UPS and an accumulation of K48-ubiquitin chains in the nucleus. Furthermore, K48-ubiquitinated lamins A and C are degraded by compensatory enhanced autophagy, as shown by increased autophagosome formation and binding of lamins A and C to microtubule-associated protein 1A/1B-light chain 3. Finally, chaperone 4-PBA augmented protein degradation by restoring UPS activity as well as autophagy in the patient cells. In summary, our results suggest that the p.S143P-mutant lamins A and C have overloading and deleterious effects on protein degradation machinery and pharmacological interventions with compounds enhancing protein degradation may be beneficial for cell homeostasis.
- Published
- 2022
25. Effect of hemin, baicalein and heme oxygenase-1 (HO-1) enzyme activity inhibitors on Cd-induced accumulation of HO-1, HSPs and aggresome-like structures in Xenopus kidney epithelial cells.
- Author
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Campbell, James H. and Heikkila, John J.
- Subjects
- *
HEMIN , *IMMUNOFLUORESCENCE , *XENOPUS , *EPITHELIAL cells , *HEME oxygenase , *EPITHELIUM , *KIDNEY diseases , *MITOCHONDRIAL membranes - Abstract
Cadmium is a highly toxic environmental pollutant that can cause many adverse effects including cancer, neurological disease and kidney damage. Aquatic amphibians are particularly susceptible to this toxicant as it was shown to cause developmental abnormalities and genotoxic effects. In mammalian cells, the accumulation of heme oxygenase-1 (HO-1), which catalyzes the breakdown of heme into CO, free iron and biliverdin, was reported to protect cells against potentially lethal concentrations of CdCl 2 . In the present study, CdCl 2 treatment of A6 kidney epithelial cells, derived from the frog, Xenopus laevis , induced the accumulation of HO-1, heat shock protein 70 (HSP70) and HSP30 as well as an increase in the production of aggregated protein and aggresome-like structures. Treatment of cells with inhibitors of HO-1 enzyme activity, tin protoporphyrin (SnPP) and zinc protoporphyrin (ZnPP), enhanced CdCl 2 -induced actin cytoskeletal disorganization and the accumulation of HO-1, HSP70, aggregated protein and aggresome-like structures. Treatment of cells with hemin and baicalein, which were previously shown to provide cytoprotection against various stresses, induced HO-1 accumulation in a concentration-dependent manner. Also, treatment of cells with hemin and baicalein suppressed CdCl 2 -induced actin dysregulation and the accumulation of aggregated protein and aggresome-like structures. This cytoprotective effect was inhibited by SnPP. These results suggest that HO-1-mediated protection against CdCl 2 toxicity includes the maintenance of actin cytoskeletal and microtubular structure and the suppression of aggregated protein and aggresome-like structures. [ABSTRACT FROM AUTHOR]
- Published
- 2018
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26. Effect of isothiocyanates, BITC and PEITC, on stress protein accumulation, protein aggregation and aggresome-like structure formation in Xenopus A6 kidney epithelial cells.
- Author
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Khamis, Imran and Heikkila, John J.
- Subjects
- *
ISOTHIOCYANATES , *HEAT shock proteins , *HEME oxygenase , *IMMUNOCYTOCHEMISTRY , *MICROTUBULES - Abstract
Numerous studies have elucidated the health benefits of organosulfur compounds, known as isothiocyanates (ITCs), derived from cruciferous vegetables. As electrophiles, ITCs have the ability to directly bind and modify thiol-containing compounds such as glutathione and cellular protein, including tubulin. While the biochemical effects of ITCs have been well characterized, less information is available regarding their effects on the accumulation of stress-inducible heme oxygenase-1 (HO-1), heat shock proteins (HSPs) and the possible formation of aggregated protein due to thiol modification. The present study has examined the effect of the ITCs, benzyl isothiocyanate (BITC) and phenethyl isothiocyanate (PEITC), on the accumulation of HO-1, HSP70 and HSP30 in Xenopus laevis A6 kidney epithelial cells. Immunoblot analysis revealed that both BITC and PEITC induced the accumulation of HO-1 and HSP70 whereas HSP30 levels were enhanced only in cells treated with BITC. Immunocytochemistry determined that ITC treatment induced F-actin disorganization and membrane ruffling and enhanced accumulation of HO-1 in the cytoplasm. Additionally, BITC induced enhanced levels of ubiquitinated protein, aggregated protein, and the collapse and fragmentation of microtubules. In comparison, treatment of cells with the proteasomal inhibitor, MG132, induced the accumulation of all three stress proteins, aggregated protein and aggresome-like structures. Finally, cells pretreated with BITC inhibited the formation of MG132-induced aggresome-like structures in the perinuclear region. This latter finding suggests that BITC-induced microtubule fragmentation may impede the movement of aggregated protein via microtubules and their subsequent coalescence into aggresome-like structures in the perinuclear region. [ABSTRACT FROM AUTHOR]
- Published
- 2018
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27. B Cells and Antibodies in Kawasaki Disease
- Author
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Michael E. Lindquist and Mark D. Hicar
- Subjects
aggresomes ,antibodies ,B cells ,plasmablasts ,inclusions ,virus-like particles ,endothelial ,Biology (General) ,QH301-705.5 ,Chemistry ,QD1-999 - Abstract
The etiology of Kawasaki disease (KD), the leading cause of acquired heart disease in children, is currently unknown. Epidemiology supports a relationship of KD to an infectious disease. Several pathological mechanisms are being considered, including a superantigen response, direct invasion by an infectious etiology or an autoimmune phenomenon. Treating affected patients with intravenous immunoglobulin is effective at reducing the rates of coronary aneurysms. However, the role of B cells and antibodies in KD pathogenesis remains unclear. Murine models are not clear on the role for B cells and antibodies in pathogenesis. Studies on rare aneurysm specimens reveal plasma cell infiltrates. Antibodies generated from these aneurysmal plasma cell infiltrates showed cross-reaction to intracellular inclusions in the bronchial epithelium of a number of pathologic specimens from children with KD. These antibodies have not defined an etiology. Notably, a number of autoantibody responses have been reported in children with KD. Recent studies show acute B cell responses are similar in children with KD compared to children with infections, lending further support of an infectious disease cause of KD. Here, we will review and discuss the inconsistencies in the literature in relation to B cell responses, specific antibodies, and a potential role for humoral immunity in KD pathogenesis or diagnosis.
- Published
- 2019
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28. Aggresomes predict poor outcomes and implicate proteostasis in the pathogenesis of pediatric choroid plexus tumors
- Author
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Ahmed A. Sayed, Noura Mahmoud, Ayda Youssef, Sameh Magdeldin, Mohamed S. Zaghloul, Mohamed Soudy, Amal Mosaab, Nada Amer, Nouran Al-Shehaby, Mohamed El-Beltagy, Shahenda El-Naggar, Hala Taha, Dina Hesham, Aya Osama, and Moatasem El-Ayadi
- Subjects
Male ,Proteomics ,Cancer Research ,Choroid Plexus Neoplasms ,Choroid plexus ,Biology ,Inclusion bodies ,Pathogenesis ,Humans ,Child ,Pathological ,Aggresomes ,Proteogenomics ,Retrospective Studies ,Inclusion Bodies ,Prognosis ,Proteostasis ,Aggresome ,Neurology ,Oncology ,Proteotoxicity ,Cancer research ,Laboratory Investigation ,Female ,Neurology (clinical) - Abstract
Purpose Protein misfolding and aggregation result in proteotoxic stress and underlie the pathogenesis of many diseases. To overcome proteotoxicity, cells compartmentalize misfolded and aggregated proteins in different inclusion bodies. The aggresome is a paranuclear inclusion body that functions as a storage compartment for misfolded proteins. Choroid plexus tumors (CPTs) are rare neoplasms comprised of three pathological subgroups. The underlying mechanisms of their pathogenesis remain unclear. This study aims to elucidate the prognostic role and the biological effects of aggresomes in pediatric CPTs. Methods We examined the presence of aggresomes in 42 patient-derived tumor tissues by immunohistochemistry and we identified their impact on patients’ outcomes. We then investigated the proteogenomics signature associated with aggresomes using whole-genome DNA methylation and proteomic analysis to define their role in the pathogenesis of pediatric CPTs. Results Aggresomes were detected in 64.2% of samples and were distributed among different pathological and molecular subgroups. The presence of aggresomes with different percentages was correlated with patients’ outcomes. The ≥ 25% cutoff had the most significant impact on overall and event-free survival (p-value Conclusions These results support the role of aggresome as a novel prognostic molecular marker for pediatric CPTs that was comparable to the molecular classification in segregating samples into two distinct subgroups, and to the pathological stratification in the prediction of patients’ outcomes. Moreover, the proteogenomic signature of CPTs displayed altered protein homeostasis, manifested by enrichment in processes related to protein quality control.
- Published
- 2021
29. Chaperones as Suppressors of Protein Misfolded Oligomer Toxicity.
- Author
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Mannini, Benedetta and Chiti, Fabrizio
- Subjects
MOLECULAR chaperones ,OLIGOMERS ,PROTEOLYSIS - Abstract
Chaperones have long been recognized to play well defined functions such as to: (i) assist protein folding and promote formation and maintenance of multisubunit complexes; (ii) mediate protein degradation; (iii) inhibit protein aggregation; and (iv) promote disassembly of undesired aberrant protein aggregates. In addition to these well-established functions, it is increasingly clear that chaperones can also interact with aberrant protein aggregates, such as pre-fibrillar oligomers and fibrils, and inhibit their toxicity commonly associated with neurodegenerative diseases without promoting their disassembly. In particular, the evidence collected so far in different labs, exploiting different experimental approaches and using different chaperones and client aggregated proteins, indicates the existence of two distinct mechanisms of action mediated by the chaperones to neutralize the toxicity of aberrant proteins oligomers: (i) direct binding of the chaperones to the hydrophobic patches exposed on the oligomer/fibril surface, with resulting shielding or masking of the moieties responsible for the aberrant interactions with cellular targets; (ii) chaperone-mediated conversion of aberrant protein aggregates into large and more innocuous species, resulting in a decrease of their surface-to-volume ratio and diffusibility and in deposits more easily manageable by clearance mechanisms, such as autophagy. In this review article we will describe the in vitro and in vivo evidence supporting both mechanisms and how this results in a suppression of the detrimental effects caused by protein misfolded aggregates. [ABSTRACT FROM AUTHOR]
- Published
- 2017
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30. Characterization of cadmium chloride-induced BiP accumulation in Xenopus laevis A6 kidney epithelial cells.
- Author
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Shirriff, Cody S. and Heikkila, John J.
- Subjects
- *
CADMIUM , *XENOPUS , *EPITHELIAL cells , *MOLECULAR genetics , *GENOMES - Abstract
Endoplasmic reticulum (ER) stress can result in the accumulation of unfolded/misfolded protein in the ER lumen, which can trigger the unfolded protein response (UPR) resulting in the activation of various genes including immunoglobulin-binding protein (BiP; also known as glucose-regulated protein 78 or HSPA5). BiP, an ER heat shock protein 70 (HSP70) family member, binds to unfolded protein, inhibits their aggregation and re-folds them in an ATP-dependent manner. While cadmium, an environmental contaminant, was shown to induce the accumulation of HSP70 in vertebrate cells, less information is available regarding the effect of this metal on BiP accumulation or function. In this study, cadmium chloride treatment of Xenopus laevis A6 kidney epithelial cells induced a dose- and time-dependent increase in BiP, HSP70 and heme oxygenase-1 (HO-1) accumulation. Exposure of cells to a relatively low cadmium concentration at a mild heat shock temperature of 30 °C greatly enhanced BiP and HSP70 accumulation compared to cadmium at 22 °C. Treatment of cells with the glutathione synthesis inhibitor, buthionine sulfoximine, enhanced cadmium-induced BiP and HSP70 accumulation. Immunocytochemistry revealed that cadmium-induced BiP accumulation occurred in a punctate pattern in the perinuclear region. In some cells treated with cadmium chloride or the proteasomal inhibitor, MG132, large BiP complexes were observed that co-localized with aggregated protein or aggresome-like structures. These BiP/aggresome-like structures were also observed in cells treated simultaneously with cadmium at 30 °C or in the presence of buthionine sulfoximine. In amphibians, the association of BiP with unfolded protein and its possible role in aggresome function may be vital in the maintenance of cellular proteostasis. [ABSTRACT FROM AUTHOR]
- Published
- 2017
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31. The expression and function of hsp30-like small heat shock protein genes in amphibians, birds, fish, and reptiles.
- Author
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Heikkila, John J.
- Subjects
- *
HEAT shock proteins , *GENE expression , *MOLECULAR chaperones , *HOMEOSTASIS , *ANIMAL species , *PROTEIN folding - Abstract
Small heat shock proteins (sHSPs) are a superfamily of molecular chaperones with important roles in protein homeostasis and other cellular functions. Amphibians, reptiles, fish and birds have a shsp gene called hsp30 , which was also referred to as hspb11 or hsp25 in some fish and bird species. Hsp30 genes, which are not found in mammals, are transcribed in response to heat shock or other stresses by means of the heat shock factor that is activated in response to an accumulation of unfolded protein. Amino acid sequence analysis revealed that representative HSP30s from different classes of non-mammalian vertebrates were distinct from other sHSPs including HSPB1/HSP27. Studies with amphibian and fish recombinant HSP30 determined that they were molecular chaperones since they inhibited heat- or chemically-induced aggregation of unfolded protein. During non-mammalian vertebrate development, hsp30 genes were differentially expressed in selected tissues. Also, heat shock-induced stage-specific expression of hsp30 genes in frog embryos was regulated at the level of chromatin structure. In adults and/or tissue culture cells, hsp30 gene expression was induced by heat shock, arsenite, cadmium or proteasomal inhibitors, all of which enhanced the production of unfolded/damaged protein. Finally, immunocytochemical analysis of frog and chicken tissue culture cells revealed that proteotoxic stress-induced HSP30 accumulation co-localized with aggresome-like inclusion bodies. The congregation of damaged protein in aggresomes minimizes the toxic effect of aggregated protein dispersed throughout the cell. The current availability of probes to detect the presence of hsp30 mRNA or encoded protein has resulted in the increased use of hsp30 gene expression as a marker of proteotoxic stress in non-mammalian vertebrates. [ABSTRACT FROM AUTHOR]
- Published
- 2017
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32. Upregulation of brain cholesterol levels inhibits mitophagy in Alzheimer disease
- Author
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Anna Colell, Xenia Abadin, Cristina de Dios, Vicente Roca-Agujetas, Fundació La Marató de TV3, Ministerio de Ciencia, Innovación y Universidades (España), and Agencia Estatal de Investigación (España)
- Subjects
0301 basic medicine ,Amyloid beta ,PINK1 ,Mitochondrion ,Parkin ,03 medical and health sciences ,Mitophagy ,Autophagy ,medicine ,Molecular Biology ,Aggresomes ,Optineurin ,030102 biochemistry & molecular biology ,biology ,Cell Biology ,medicine.disease ,Cell biology ,Cholesterol ,030104 developmental biology ,biology.protein ,Alzheimer disease ,Alzheimer's disease - Abstract
Mitochondrial dysfunction is behind several neurodegenerative diseases, including Alzheimer disease (AD). Accumulation of damaged mitochondria is already observed at the early stages of AD and has been linked to impaired mitophagy, but the mechanisms underlying this alteration are still not fully known. In our recent study, we show that intracellular cholesterol enrichment can downregulate amyloid beta (Aβ)-induced mitophagy. Mitochondrial glutathione depletion resulting from high cholesterol levels promotes PINK1 (PTEN induced kinase 1)-mediated mitophagosome formation; however, mitophagy flux is ultimately disrupted, most likely due to fusion deficiency of endosomes-lysosomes caused by cholesterol. Meanwhile, in APP-PSEN1-SREBF2 mice, an AD mouse model that overexpresses the cholesterol-related transcription factor SREBF2, cholesterol accumulation prompts an oxidative- and age-dependent cytosolic aggregation of the mitophagy adaptor OPTN (optineurin), which prevents mitophagosome formation despite enhanced PINK1-PRKN/parkin signaling. Hippocampal neurons from postmortem brain of AD individuals reproduce the progressive accumulation of OPTN in aggresome-like structures accompanied by high levels of mitochondrial cholesterol in advanced stages of the disease. Overall, these data provide new insights into the impairment of the PINK1-PRKN mitophagy pathway in AD and suggest the combination of mitophagy inducers with strategies focused on restoring the cholesterol homeostasis and mitochondrial redox balance as a potential disease-modifying therapy for AD., This work was supported by the Fundació la Marató de TV3 [2014-093]; Ministerio de Ciencia, Innovación y Universidades (ES) [RTI2018- 095572-B-100].
- Published
- 2021
33. Proteasome-Rich PaCS as an Oncofetal UPS Structure Handling Cytosolic Polyubiquitinated Proteins. In Vivo Occurrence, in Vitro Induction, and Biological Role
- Author
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Enrico Solcia, Vittorio Necchi, Patrizia Sommi, and Vittorio Ricci
- Subjects
proteasome ,polyubiquitinated proteins ,heat-shock proteins ,PaCS ,neoplastic cells ,fetal cells ,microbial oncogenic proteins ,trophic factors/interleukins ,sequestosomes ,aggresomes ,Biology (General) ,QH301-705.5 ,Chemistry ,QD1-999 - Abstract
In this article, we outline and discuss available information on the cellular site and mechanism of proteasome interaction with cytosolic polyubiquitinated proteins and heat-shock molecules. The particulate cytoplasmic structure (PaCS) formed by barrel-like particles, closely reproducing in vivo the high-resolution structure of 26S proteasome as isolated in vitro, has been detected in a variety of fetal and neoplastic cells, from living tissue or cultured cell lines. Specific trophic factors and interleukins were found to induce PaCS during in vitro differentiation of dendritic, natural killer (NK), or megakaryoblastic cells, apparently through activation of the MAPK-ERK pathway. Direct interaction of CagA bacterial oncoprotein with proteasome was shown inside the PaCSs of a Helicobacter pylori-infected gastric epithelium, a finding suggesting a role for PaCS in CagA-mediated gastric carcinogenesis. PaCS dissolution and autophagy were seen after withdrawal of inducing factors. PaCS-filled cell blebs and ectosomes were found in some cells and may represent a potential intercellular discharge and transport system of polyubiquitinated antigenic proteins. PaCS differs substantially from the inclusion bodies, sequestosomes, and aggresomes reported in proteinopathies like Huntington or Parkinson diseases, which usually lack PaCS. The latter seems more linked to conditions of increased cell proliferation/differentiation, implying an increased functional demand to the ubiquitin–proteasome system.
- Published
- 2018
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34. Defective transcription elongation in human cancers imposes targetable proteotoxic vulnerability
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B. Muhammad, L.G. Parks, K. Komurov, and L.M. Privette Vinnedge
- Subjects
Cancer Research ,Transcription elongation defects (TEdef) ,Oncology ,Autophagy ,Neoplasms. Tumors. Oncology. Including cancer and carcinogens ,Protein homeostasis ,Proteotoxic stress ,RC254-282 ,Original Research ,Aggresomes - Abstract
Highlights • Transcription elongation defects (TEdef), which occur in approximately 20% of breast cancers, are identified by high levels of spurious transcripts, intron retention, and loss of gene body exon expression. • There is transcriptional upregulation of protein homeostasis pathway genes in TEdef tumors. • TEdef breast cancer cells demonstrate high levels of ubiquitinated insoluble proteins and protein aggregates. • TEdef breast cancers show evidence of upregulated autophagy and are more sensitive to autophagy inhibitors than cancer cells with proficient transcription elongation (TEprof)., Successful cancer therapy is contingent on identifying cancer-specific aberrant phenotypes and their associated vulnerabilities. We recently reported that a subset of almost every cancer type contains a genome-wide defect in RNA Polymerase II-mediated transcription elongation (TEdef), which impairs the expression of long genes and confers resistance to anti-tumor immune attack. Using a combination of computational analysis and laboratory experiments, we report that tumor cells with TEdef have widespread overexpression of the components of the protein homeostasis machinery (mostly composed of short genes), including protein folding and clearance. Accordingly, TEdef cells were characterized by abnormally high levels of insoluble protein aggregates in the cytoplasm and autophagy influx. We present evidence that TEdef cells exhibit impaired clearance of misfolded protein aggregates through the ubiquitin-proteasome system, and thus rely on autophagy for their degradation. As such, while these cells were highly resistant to proteasome inhibitors, they were acutely sensitive to inhibitors of autophagy in vitro and in vivo. This study reveals a major aberrant phenotype that is observed in ∼15–25% of all cancers and characterizes a unique cellular vulnerability that can be readily exploited in the clinic to improve treatment efficacy.
- Published
- 2021
35. The formation of peripheral myelin protein 22 aggregates is hindered by the enhancement of autophagy and expression of cytoplasmic chaperones
- Author
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Jenny Fortun, Jonathan D. Verrier, Jocelyn C. Go, Irina Madorsky, William A. Dunn, and Lucia Notterpek
- Subjects
Autophagosome ,Proteasome ,Neuropathy ,Schwann cells ,Protein misfolding ,Aggresomes ,Neurosciences. Biological psychiatry. Neuropsychiatry ,RC321-571 - Abstract
The accumulation of misfolded proteins is associated with various neurodegenerative conditions. Peripheral myelin protein 22 (PMP22) is a hereditary neuropathy-linked, short-lived molecule that forms aggresomes when the proteasome is inhibited or the protein is mutated. We previously showed that the removal of pre-existing PMP22 aggregates is assisted by autophagy. Here we examined whether the accumulation of such aggregates could be suppressed by experimental induction of autophagy and/or chaperones. Enhancement of autophagy during proteasome inhibition hinders protein aggregate formation and correlates with a reduction in accumulated proteasome substrates. Conversely, simultaneous inhibition of autophagy and the proteasome augments the formation of aggregates. An increase of heat shock protein levels by geldanamycin treatment or heat shock preconditioning similarly hampers aggresome formation. The beneficial effects of autophagy and chaperones in preventing the accumulation of misfolded PMP22 are additive and provide a potential avenue for therapeutic approaches in hereditary neuropathies linked to PMP22 mutations.
- Published
- 2007
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36. Eukaryotic aggresomes: from a model of conformational diseases to an emerging type of immobilized biocatalyzers.
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Corchero, José
- Subjects
- *
PROTEIN conformation , *EUKARYOTES , *BIOCATALYSIS , *DRUG delivery systems , *PROTEOLYSIS , *BIOTECHNOLOGY - Abstract
Unraveling the characteristics and putative applications of naturally occurring protein aggregates has received an increasing interest during the last years. For example, the finding that the proteins embedded within bacterial inclusion bodies are, at least partially, biologically functional opened new opportunities for their rational design and application as naturally self-immobilized biocatalysts or as new drug delivery systems ('nanopills'). In another scenario, it is well established that 'conformational diseases' are caused by misfolding and protein aggregation in different cells and tissues. The presence of such protein aggregates is a hallmark of these conditions, therefore becoming an excellent target for new therapeutic approaches for such devastating pathologies. Aggresomes are protein aggregates found in eukaryotic cells when the intracellular protein degradation machinery is overtitered. These protein-based nanoparticles are increasingly becoming excellent models in studies aimed to obtain a better understanding and control over protein aggregation processes in eukaryotic cells. In this work, we focus on some of the latest findings in the field of putative aggresome applications in biotechnology, as a new type of self-assembled immobilized biocatalysts, and in nanomedicine, mainly on their relationship with conformational diseases and the rational design of better therapeutics through a deeper understanding of protein aggregation processes. [ABSTRACT FROM AUTHOR]
- Published
- 2016
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37. Host cell targets for African swine fever virus.
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Muñoz-Moreno, Raquel, Galindo, Inmaculada, Cuesta-Geijo, Miguel Ángel, Barrado-Gil, Lucía, and Alonso, Covadonga
- Subjects
- *
AFRICAN swine fever virus , *INTRACELLULAR pathogens , *DNA viruses , *ENDOCYTOSIS , *HOST-virus relationships - Abstract
Viruses are strict intracellular pathogens that require the cellular environment to complete a successful infection. Among them, African swine fever virus (ASFV) is an evolutionary ancient DNA virus, endemic in Africa, which is nowadays causing an emergent disease in Europe with a potential high economic impact in the pig industry. It is well known that host-cell components are critical crossroads mapping the virus path for a productive infection, some of them at the endocytic pathway. Considering that ASFV infectious cycle strongly relies in several factors from the host cell, the study of virus–host interactions remains crucial as they will reveal the obstacles, routes and tracks, hints and the target waypoint in the virus journey to destination. [ABSTRACT FROM AUTHOR]
- Published
- 2015
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- View/download PDF
38. Quality control in the apoA-I secretory pathway
- Author
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Shaila Bhat, Manal Zabalawi, Mark C. Willingham, Gregory S. Shelness, Michael J. Thomas, and Mary G. Sorci-Thomas
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apolipoprotein A-I ,high density lipoprotein ,mutant apolipoprotein A-I ,protein secretion ,quality control pathway ,aggresomes ,Biochemistry ,QD415-436 - Abstract
From a total of 47 known apolipoprotein A-I (apoA-I) mutations, only 18 are linked to low plasma HDL apoA-I concentrations, and 78% of these map to apoA-I helices 6 and 7 (residues 143–186). Gene transfer and transgenic mouse studies have shown that several helix 6 apoA-I mutations have reduced hepatic HDL production. Our objective was to examine the impact of helix 6 modifications on intracellular biosynthetic processing and secretion of apoA-I. Cells were transfected with wild-type or mutant apoA-I, radiolabeled with [35S]Met/Cys, and then placed in unlabeled medium for up to 4 h. Results show that >90% of newly synthesized wild-type apoA-I was secreted by 60 min. Over the same length of time, only 20% of helix 6 deletion mutant (Δ6 apoA-I) was secreted, whereas 80% remained cell associated. Microscopic and biochemical studies revealed that cell-associated Δ6 apoA-I was located predominantly within the cytoplasm as lipid-protein inclusions, whereas wild-type apoA-I was localized in the endoplasmic reticulum/Golgi. Results using other helix deletions or helix 6 substitution mutations indicated that only complete removal of helix 6 resulted in massive cytoplasmic accumulation.These data suggest that alterations in native apoA-I conformation can lead to aberrant trafficking and accumulation of apolipoprotein-phospholipid structures. Thus, conformation-dependent alterations in intracellular trafficking and turnover may underlie the reduced plasma HDL concentrations observed in individuals harboring deletion mutations within helix 6.
- Published
- 2004
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39. Aggresome Formation in Neuropathy Models Based on Peripheral Myelin Protein 22 Mutations
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Mary C. Ryan, Eric M. Shooter, and Lucia Notterpek
- Subjects
aggresomes ,PMP22 ,ubiquitin-proteasome pathway ,Trembler mice ,protein degradation ,Schwann cells ,Neurosciences. Biological psychiatry. Neuropsychiatry ,RC321-571 - Abstract
Alterations in peripheral myelin protein 22 (PMP22) gene expression are associated with demyelinating peripheral neuropathies. Overexpression of wild type (wt) PMP22 or inhibition of proteasomal degradation lead to the formation of aggresomes, intracellular ubiquitinated PMP22 aggregates. Aggresome formation has now been observed with two mutant PMP22s, the Tr- and TrJ-PMP22 when the proteasome is inhibited. The formation of these aggresomes required intact microtubules and involved the recruitment of chaperones, including Hsp40, Hsp70, and αB-crystallin. Spontaneously formed ubiquitinated PMP22 aggregates were also observed in Schwann cells of homozygous TrJ mice. Significant upregulation of both the ubiquitin-proteasomal and lysosomal pathways occurred in affected nerves suggesting that two pathways of PMP22 degradation are present. Thus, the presence of aggresomes appears to be a common finding in neuropathy models of PMP22 overexpression and of some point mutations known to cause neuropathy in mice and humans.
- Published
- 2002
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40. Extensive accumulation of misfolded protein aggregates during natural aging and senescence.
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Cuanalo-Contreras K, Schulz J, Mukherjee A, Park KW, Armijo E, and Soto C
- Abstract
Accumulation of misfolded protein aggregates is a hallmark event in many age-related protein misfolding disorders, including some of the most prevalent and insidious neurodegenerative diseases. Misfolded protein aggregates produce progressive cell damage, organ dysfunction, and clinical changes, which are common also in natural aging. Thus, we hypothesized that aging is associated to the widespread and progressive misfolding and aggregation of many proteins in various tissues. In this study, we analyzed whether proteins misfold, aggregate, and accumulate during normal aging in three different biological systems, namely senescent cells, Caenorhabditis elegans , and mouse tissues collected at different times from youth to old age. Our results show a significant accumulation of misfolded protein aggregates in aged samples as compared to young materials. Indeed, aged samples have between 1.3 and 2.5-fold (depending on the biological system) higher amount of insoluble proteins than young samples. These insoluble proteins exhibit the typical characteristics of disease-associated aggregates, including insolubility in detergents, protease resistance, and staining with amyloid-binding dye as well as accumulation in aggresomes. We identified the main proteins accumulating in the aging brain using proteomic studies. These results show that the aged brain contain large amounts of misfolded and likely non-functional species of many proteins, whose soluble versions participate in cellular pathways that play fundamental roles in preserving basic functions, such as protein quality control, synapsis, and metabolism. Our findings reveal a putative role for protein misfolding and aggregation in aging., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Cuanalo-Contreras, Schulz, Mukherjee, Park, Armijo and Soto.)
- Published
- 2023
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41. A novel bio-functional material based on mammalian cell aggresomes.
- Author
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Rodríguez-Carmona, Escarlata, Mendoza, Rosa, Ruiz-Cánovas, Eugènia, Ferrer-Miralles, Neus, Abasolo, Ibane, Schwartz, Simó, Villaverde, Antonio, and Corchero, José
- Subjects
- *
MAMMALIAN cell cycle , *PROTEOLYSIS , *GALACTOSIDASES , *RECOMBINANT proteins , *NANOPARTICLES , *CELLULAR inclusions - Abstract
Aggresomes are protein aggregates found in mammalian cells when the intracellular protein degradation machinery is over-titered. Despite that they abound in cells producing recombinant proteins of biomedical and biotechnological interest, the physiological roles of these protein clusters and the functional status of the embedded proteins remain basically unexplored. In this work, we have determined for the first time that, like in bacterial inclusion bodies, deposition of recombinant proteins into aggresomes does not imply functional inactivation. As a model, human α-galactosidase A (GLA) has been expressed in mammalian cells as enzymatically active, mechanically stable aggresomes showing higher thermal stability than the soluble GLA version. Since aggresomes are easily produced and purified, we propose these particles as novel functional biomaterials with potential as carrier-free, self-immobilized catalyzers in biotechnology and biomedicine. [ABSTRACT FROM AUTHOR]
- Published
- 2015
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42. Role of Cigarette Smoke-Induced Aggresome Formation in Chronic Obstructive Pulmonary Disease-Emphysema Pathogenesis.
- Author
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Tran, Ian, Changhoon Ji, Ni, Inzer, Taehong Min, Danni Tang, and Vij, Neeraj
- Published
- 2015
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43. Intensified autophagy compromises the efficacy of radiotherapy against prostate cancer.
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Koukourakis, Michael I., Kalamida, Dimitra, Mitrakas, Achilleas, Pouliliou, Stamatia, Kalamida, Sofia, Sivridis, Efthimios, and Giatromanolaki, Alexandra
- Subjects
- *
PROSTATE cancer treatment , *AUTOPHAGY , *CANCER radiotherapy , *PROSTATECTOMY , *TREATMENT effectiveness - Abstract
Introduction Radiotherapy is an equivalent alternative or complement to radical prostatectomy, with high therapeutic efficacy. High risk patients, however, experience high relapse rates, so that research on radio-sensitization is the most evident route to improve curability of this common disease. Materials and methods In the current study we investigated the autophagic activity in a series of patients with localized prostate tumors treated with radical radiotherapy, using the LC3A and the LAMP2a proteins as markers of autophagosome and lysosome cellular content, respectively. The role of autophagy on prostate cancer cell line resistance to radiation was also examined. Results Using confocal microscopy on tissue biopsies, we showed that prostate cancer cells have, overall, high levels of LC3A and low levels of LAMP2a compared to normal prostate glands. Tumors with a ‘highLC3A/lowLAMP2a’ phenotype, suggestive of intensified lysosomal consumption, had a significantly poorer biochemical relapse free survival. The PC3 radioresistant cell line sustained remarkably its autophagic flux ability after radiation, while the DU145 radiosensitive one experiences a prolonged blockage of the autophagic process. This was assessed with aggresome accumulation detection and LC3A/LAMP2a double immunofluorescence, as well as with sequestrosome/p62 protein detection. By silencing the LC3A or LAMP2a expression, both cell lines became more sensitive to escalated doses of radiation. Conclusions High base line autophagy activity and cell ability to sustain functional autophagy define resistance of prostate cancer cells to radiotherapy. This can be reversed by blocking up-regulated components of the autophagy pathway, which may prove of importance in the field of clinical radiotherapy. [ABSTRACT FROM AUTHOR]
- Published
- 2015
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- View/download PDF
44. SGTA regulates the cytosolic quality control of hydrophobic substrates.
- Author
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Wunderley, Lydia, Leznicki, Pawel, Payapilly, Aishwarya, and High, Stephen
- Subjects
- *
HYDROPHOBIC compounds , *POLYPEPTIDES , *CYTOSOL , *UBIQUITINATION , *MEMBRANE proteins , *DENATURATION of proteins , *GLUTAMINE , *MOLECULAR chaperones - Abstract
Hydrophobic amino acids are normally shielded from the cytosol and their exposure is often used as an indicator of protein misfolding to enable the chaperone-mediated recognition and quality control of aberrant polypeptides. Mislocalised membrane proteins (MLPs) represent a particular challenge to cellular quality control, and, in this study, membrane protein fragments have been exploited to study a specialised pathway that underlies the efficient detection and proteasomal degradation of MLPs. Our data show that the BAG6 complex and SGTA compete for cytosolic MLPs by recognition of their exposed hydrophobicity, and the data suggest that SGTA acts to maintain these substrates in a non-ubiquitylated state. Hence, SGTA might counter the actions of BAG6 to delay the ubiquitylation of specific precursors and thereby increase their opportunity for successful post-translational delivery to the endoplasmic reticulum. However, when SGTA is overexpressed, the normally efficient removal of aberrant MLPs is delayed, increasing their steady-state level and promoting aggregation. Our data suggest that SGTA regulates the cellular fate of a range of hydrophobic polypeptides should they become exposed to the cytosol. [ABSTRACT FROM AUTHOR]
- Published
- 2014
- Full Text
- View/download PDF
45. A novel bio-functional material based on mammalian cell aggresomes
- Subjects
Protein nanoparticles ,Immobilized catalyzer ,Recombinant protein ,Aggresomes - Published
- 2021
46. The c10orf10 gene product is a new link between oxidative stress and autophagy.
- Author
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Stepp, Marcus W., Folz, Rodney J., Yu, Jerry, and Zelko, Igor N.
- Subjects
- *
AUTOPHAGY , *PROGESTERONE , *HYPOXEMIA , *SUPEROXIDE dismutase , *MESSENGER RNA , *GENE expression - Abstract
Abstract: The human c10orf10 gene product, also known as decidual protein induced by progesterone (DEPP), is known to be differentially regulated in mouse tissues in response to hypoxia and oxidative stress, however its biological function remains unknown. We found that mice lacking extracellular superoxide dismutase (EC-SOD) show attenuated expression of DEPP in response to acute hypoxia. DEPP mRNA levels, as well as the activity of a reporter gene expressed under the control of the DEPP 5′-flanking region, were significantly upregulated in Hep3B and Vero cells overexpressing EC-SOD. Subcellular fractionation and immunofluorescent microscopy indicated that overexpressed DEPP is co-localized with both protein aggregates and aggresomes. Further biochemical characterization indicates that DEPP protein is unstable and undergoes rapid degradation. Inhibition of proteasome activities significantly increases DEPP protein levels in soluble and insoluble cytosolic fractions. Attenuation of autophagosomal activity by 3-methyladenine increases DEPP protein levels while activation of autophagy by rapamycin reduced DEPP protein levels. In addition, ectopic overexpression of DEPP leads to autophagy activation, while silencing of DEPP attenuates autophagy. Collectively, these results indicate that DEPP is a major hypoxia-inducible gene involved in the activation of autophagy and whose expression is regulated by oxidative stress. [Copyright &y& Elsevier]
- Published
- 2014
- Full Text
- View/download PDF
47. Ubiquilin-1 immunoreactivity is concentrated on Hirano bodies and dystrophic neurites in Alzheimer's disease brains.
- Author
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Satoh, J., Tabunoki, H., Ishida, T., Saito, Y., and Arima, K.
- Subjects
- *
ADAPTOR proteins , *DYSTROPHY , *ALZHEIMER'S disease , *CARCINOGENESIS , *IMMUNOHISTOCHEMISTRY - Abstract
Aims Ubiquilin-1 acts as an adaptor protein that mediates the translocation of polyubiquitinated proteins to the proteasome for degradation. Although previous studies suggested a key role of ubiquilin-1 in the pathogenesis of Alzheimer's disease ( AD), a direct relationship between ubiquilin-1 and Hirano bodies in AD brains remains unknown. Methods By immunohistochemistry, we studied ubiquilin-1 and ubiquilin-2 expression in the frontal cortex and the hippocampus of six AD and 13 control cases. Results Numerous Hirano bodies, accumulated in the hippocampal CA1 region of AD brains, expressed intense immunoreactivity for ubiquilin-1. They were much less frequently found in control brains. However, Hirano bodies did not express a panel of markers for proteasome, autophagosome or pathogenic proteins, such as ubiquilin-2, ubiquitin, p62, LC3, beclin-1, HDAC6, paired helical filament ( PHF)-tau, protein-disulphide isomerase ( PDI) and phosphorylated TDP-43, but some of them expressed C9orf72. Ubiquilin-1-immunoreactive deposits were classified into four distinct morphologies, such as rod-shaped structures characteristic of Hirano bodies, dystrophic neurites contacting senile plaques, fragmented structures accumulated in the lesions affected with severe neuronal loss, and thread-shaped structures located mainly in the molecular layer of the hippocampus. Conclusions Ubiquilin-1 immunoreactivity is concentrated on Hirano bodies and dystrophic neurites in AD brains, suggesting that aberrant expression of ubiquilin-1 serves as one of pathological hallmarks of AD. [ABSTRACT FROM AUTHOR]
- Published
- 2013
- Full Text
- View/download PDF
48. Crosstalk between autophagy and proteasome protein degradation systems: possible implications for cancer therapy.
- Author
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Wojcik, Slawomir
- Subjects
AUTOPHAGY ,UBIQUITIN ,BIODEGRADATION ,EUKARYOTIC cells ,PROTEASOME inhibitors ,BORTEZOMIB ,INFLAMMATION ,CANCER treatment ,THERAPEUTICS - Abstract
The Ubiquitin-Proteasomes System (UPS) and autophagy, two main intracellular protein degradation pathways within the eukaryotic cells which were originally regarded as rather independent, seem to be very closely related. Proteasome inhibitors, including the multipathway inhibitor bortezomib, are drawing increased attention for their therapeutic potential in the treatment of chronic inflammation and cancer, especially tumours with a high degree of malignancy. The over-activation of autophagy induces cell death and may act as a powerful tumour-suppressing mechanism. However, autophagy, serving as an important mechanism to generate nutrients in time of cellular stresses, may directly contribute to the survival of cells treated with proteasome inhibitors, and in consequence, may decrease the effectiveness of therapy. Results of studies performed on several cancer cell lines demonstrated synergy between proteasome inhibitors and autophagy inhibitors. Those results became the base for ongoing clinical trials investigating autophagy inhibition in combination with anti-cancer therapies, including bortezomib. This review provides summary of the latest data on the functioning of the UPS and the mechanisms of autophagy. The new insights describing the main pathways of autophagy activation in response to UPS inhibition related to: (i) Unfolded Protein Response, (ii) PI3K/Akt/mTOR pathway, and (iii) formation of aggresomes, are discussed. It is concluded that concomitant inhibition of the two main cellular protein degradation systems may provide new therapeutic modalities for cancer treatment. [ABSTRACT FROM AUTHOR]
- Published
- 2013
- Full Text
- View/download PDF
49. Alterações morfológicas e agregação proteica no envelhecimento da glândula mamária e no cancro da mama
- Author
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Figueira, Daniela Filipa Duarte, Helguero, Luisa, Gama, Adelina, and Oliveira, Paula Alexandra Martins de
- Subjects
Unfolded protein response ,Aging ,Protein damage ,Breast cancer ,Adaptative stress responses ,Mammary gland ,Proteostasis ,Aggresomes - Abstract
Breast cancer risk increases with aging. Aberrations occurring in the mammary cell differentiation process increase the risk of breast cancer. In general, cell aging is associated with increased protein damage which can result in toxic intra – and extracellular protein aggregates and influence cell survival. Consequently, the cells activate a series of adaptive stress responses to resolve this potentially lethal toxicity. However, with aging cells appear to become less efficient as protein aggregation in different biological systems has been reported to increase with age. Protein aggregation is associated to human diseases which have higher incidence in the aging population and are related to cell degeneration and death, such as Alzheimer’s. Little information exists regarding how human cells from different embryonic origin and differentiation stage resolve toxic protein aggregates and how this can influence carcinogenesis. Since the mammary gland develops after birth through differentiation of adult mammary stem and progenitor cells, we hypothesized that the different cell hierarchies found in the mammary tissue deal differently with age-related protein aggregation. This in turn would make certain mammary cell types more prone to suffer alterations during differentiation. This work has two objectives: 1) the study of differentiation markers in the mammary epithelium throughout aging; and 2) the evaluation of the protein aggregates observed by fluorescence in human breast cancer cases to assess the changes in proteostasis before and after hormone therapy. For objective 1, the experimental model consisted of tissue samples from C57BL/6 mice aged 1 to 29 months; and human samples of hormone therapy resistant breast tumors (samples before and after treatment) for objective 2. Methods included histological evaluation of the mouse mammary gland during aging, namely constitution and architecture of the gland and immunohistochemistry to evaluate expression of epithelial markers and mammary gland differentiation (such as CK5, CK8, ER and PR); and for objective 2, it was necessary to optimize the protocol with ProteoStat® for histological samples. Objective 1 was not achieved, possibly due to the fixation artifacts found in the samples, which made the immunohistochemical study impossible. In Objective 2, opposing the expectations, more protein aggregates were detected in tumor samples after hormone therapy, and further studies will be needed to discriminate intra and extracellular aggregates. O risco de cancro da mama aumenta com a idade. Alterações durante a diferenciação celular mamária aumentam o risco de cancro da mama. O envelhecimento celular está associado ao aumento do dano proteico, o que pode resultar em agregados proteicos com toxicidade intra e extracelular que influenciam a sobrevivência celular. Consequentemente, as células ativam uma série de respostas ao stress para responder a esta toxicidade potencialmente letal. Contudo, com o envelhecimento, as células aparentam ser menos eficientes, uma vez que tem sido descrita a agregação de proteínas em diversos sistemas biológicos com o envelhecimento. Esta agregação proteica está associada com várias doenças humanas que apresentam elevada incidência na população envelhecida e está relacionada com degeneração e morte, tal como a doença de Alzheimer. Existe pouca informação relativamente aos mecanismos utilizados pelas células humanas de diferentes origens embrionárias e estadios de diferenciação relativamente à eliminação destes agregados, bem como de que forma estes influenciam a carcinogénese. Desde que a glândula mamária se desenvolve e diferencia após o nascimento a partir de células estaminais progenitoras, colocámos a hipótese de diferentes tipos celulares reagirem de forma diferente aos agregados proteicos relacionados com o envelhecimento. Assim, alguns tipos de células mamárias podem ser mais suscetíveis a alterações durante a diferenciação. Este trabalho tem dois objetivos: 1) O estudo de marcadores de diferenciação no epitélio mamário ao longo do envelhecimento; e 2) Avaliação dos agregados proteicos observados através de fluorescência nos casos de cancro da mama humanos, para avaliar se existem alterações na proteostase antes e depois da terapia hormonal. Para o objetivo 1, o modelo experimental consistiu em amostras de tecido de ratinhos C57BL/6 com idades compreendidas entre 1 e 29 meses de idade; e amostras humanas de tumores mamários resistentes à terapia hormonal (amostras antes e após o tratamento) para o objetivo 2. Os métodos incluíram avaliação histológica da glândula mamária do ratinho durante o envelhecimento, nomeadamente constituição e arquitetura da glândula e imunohistoquímica para avaliar a expressão de marcadores epiteliais e de diferenciação da glândula mamária (tais como CK5, CK8, ER e PR); e para o objetivo 2, foi necessário a otimização do protocolo com o ProteoStat® para amostras histológicas. O objetivo 1 não foi cumprido, possivelmente devido artefactos de fixação encontrados nas amostras, que impossibilitaram o estudo imunohistoquímico. No objetivo 2, contrariamente ao esperado, foram detetados mais agregados proteicos nas amostras de tumores após a terapia hormonal, sendo necessários mais estudos para discriminar os agregados intra e extracelulares. Mestrado em Biomedicina Molecular
- Published
- 2019
50. Autophagy in asthma and chronic obstructive pulmonary disease.
- Author
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Barnes PJ, Baker J, and Donnelly LE
- Subjects
- Autophagy, Cellular Senescence, Humans, Mitophagy, Asthma drug therapy, Pulmonary Disease, Chronic Obstructive metabolism
- Abstract
Autophagy (or macroautophagy) is a key cellular process that removes damaged molecules (particularly proteins) and subcellular organelles to maintain cellular homeostasis. There is growing evidence that abnormalities in autophagy may contribute to the pathogenesis of many chronic diseases, including asthma and chronic obstructive pulmonary disease (COPD). In asthma, increased autophagy plays a role in promoting type 2 immune responses and eosinophilic inflammation, whereas decreased autophagy may be important in neutrophilic asthma. Acute exposure to cigarette smoke may activate autophagy, resulting in ciliary dysfunction and death of airway epithelial cells, whereas in stable COPD most studies have demonstrated an impairment in autophagy, with reduced autophagic flux and accumulation of abnormal mitochondria (defective mitophagy) and linked to cellular senescence. Autophagy may be increased or decreased in different cell types and depending on the cellular environment, making it difficult to target autophagy therapeutically. Several existing drugs may activate autophagy, including rapamycin, metformin, carbamazepine, cardiac glycosides and statins, whereas others, such as chloroquine, inhibit this process. However, these drugs are nonspecific and more selective drugs are now in development, which may prove useful as novel agents to treat asthma and COPD in the future., (© 2022 The Author(s).)
- Published
- 2022
- Full Text
- View/download PDF
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