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Rapid Isolation of Lysosomes from Cultured Cells Using a Twin Strep Tag
Published in Bruno Gasnier, Michael X. Zhu, Ion and Molecule Transport in Lysosomes, 2020
Jian Xiong, Jingquan He, Michael X. Zhu, Guangwei Du
Note: If the proteins of interest are multi-transmembrane proteins, the sample should not be boiled. Multi-transmembrane proteins might aggregate and fail to properly migrate in SDS-PAGE. The purity of isolated lysosomes can be determined by immunoblotting using antibodies against lysosomal proteins, such as LAMP1 and LAMP2, as well as other intracellular organelles, such as HSPA9 (mitochondria), calnexin (endoplasmic reticulum), EEA1 (early endosomes), catalase (peroxisomes) and tubulin (cytoskeleton) (Xiong et al., 2019). Only the lysosomal proteins should be detected by immunoblotting in a successful lysosome purification. To assess the changes of signalling molecules associated with lysosomes upon amino acid treatments, we measured the level of mTORC1 by immunoblotting as well, and observed decreased levels of mTOR, raptor and phosphor-mTOR (S2448) in lysosome preparations isolated from amino acid-starved cells (Xiong et al., 2019).
Micronutrients in Protecting Against Lethal Doses of Ionizing Radiation
Published in Kedar N. Prasad, Micronutrients in Health and Disease, 2019
Irradiation of retinal pigment epithelial cells (RPE cells) and human umbilical vein endothelial cells (HUVECs) increased the expression of miR-525-3p and deceased the levels of its target proteins ARRB1 (arrestin beta1) that inhibits NF-kB activity, and TNX1 (thioredoxin-1) and HSPA9 (heat shock protein family A member 9) that control cell proliferation.32 Overexpression of miR-525-3p makes cells radioresistant.
Computational characterization and integrative analysis of proteins involved in spermatogenesis
Published in C. Yan Cheng, Spermatogenesis, 2018
Pranitha Jenardhanan, Manivel Panneerselvam, Premendu P. Mathur
Among the proteins enriched in sperm tails from normozoospermic donors, TUBB2B and ODF2, which showed decreased expression in asthenozoospermic sperm tails, were found to be crucial for sperm tail motility. Tubulin beta 2B is a component of falagellar microtubule49 and outer dense fiber protein 2 forms the component of outer dense fibers, which are the accessory structures found surrounding the axenome of sperm tail and are involved in regulating elastic rigidity of sperm flagellum.50,51 Reduced expression of these proteins is obvious to impart negative effects in sperm tail movement in asthenozoospermic patients. Similarly, A- AKAP4 is the abundant structural protein in fibrous sheath that anchors cyclic-AMP-(cAMP)-dependent protein kinase A (PKA) to the fibrous sheath, therein increasing phosphorylation activities leading to activation of fibrous sheath. Deletion of AKAP4 is shown to inactivate fibrous sheath and successively impair sperm tail motility.52,53 GAPDS is another important protein that is downregulated in asthenozoospermic patients. GAPDS is located along the principal piece of sperm tail and plays a role in ATP production in the distant site of flagellum. Gene knockout studies proved that a GAPDS-deficient mouse is deficient in sperm tail movement.54 Similarly, the proteins COX6B, PHGPx, VDAC2, and HSPA2 and HSPA9 also can be used as biomarkers for understanding male infertility.
Untargeted proteomics reveals upregulation of stress response pathways during CHO-based monoclonal antibody manufacturing process leading to disulfide bond reduction
Published in mAbs, 2021
Seo-Young Park, Susan Egan, Anthony J. Cura, Kathryn L. Aron, Xuankuo Xu, Mengyuan Zheng, Michael Borys, Sanchayita Ghose, Zhengjian Li, Kyongbum Lee
Enrichment analysis showed that the proteins upregulated on day 7 in the rolled condition (clusters 4 and 5) were significantly associated with translation and metabolic processes (Tables S2 and S3). These proteins include stress-inducible protein folding and processing-related proteins located in the ER (HSP90B1, HYOU1, and RPN2), cytosol (CCT2, CCT5, and HSPCA), and mitochondria (HSPA9). In addition to chaperone function, the ER proteins (HSP90B1 and HYOU1) and heat shock proteins (HSPCA and HSPA9) also regulate Ca2+ signaling and are essential for protein assembly and folding as well as degradation of misfolded proteins.40–42 The chaperones CCT2 and CCT5 interact with heat-shock proteins (e.g., HSP90B1) to regulate Ca2+ signaling in response to stress in the ER. In CHO cells, HSP90B1 is induced by glucose limitation or ER stress.43,44 Expression of HSPA9 in CHO cells is increased with accumulation of unfolded protein in the ER.45 Recently, Xie et al.39 highlighted HSPA9 as a marker of oxidative stress in CHO cells. Upregulation of HYOU1 occurs under hypoxia,46 as well as ER stress-induced unfolded protein response. Wei et al.47 showed that these ER chaperones were upregulated as cells aged during extended CHO cell culture.
Mitochondrial autophagosomes as a mechanism of drug resistance in breast carcinoma
Published in Ultrastructural Pathology, 2018
Ayman N. Abunimer, Heba Mohammed, Katherine L. Cook, David R. Soto-Pantoja, Maria Mercedes Campos, Mones S. Abu-Asab
MKT-077 is a cytotoxic compound to a variety of cancerous cells.8,9 It affects cells through accumulating specifically within mitochondria, and subsequently disrupting mitochondrial membranes, DNA, and oxidative functions.10–14 MKT-077 also increases mitochondrial fragmentation by inhibiting mortalin (GRP75; HSPA9) in a Drp1-dependent manner.14 Mitochondria possess high negative-internal transmembrane potential which renders them a target for lipophilic cations, such as rhodamines. The intramitochondrial concentration of these compounds tends to be 100- to 1000-folds higher than the rest of the cell.15–17 MKT-077 adversely affects breast (MFC-7), colon (CX-1), melanoma (LOX), pancreas (CRL1420), and transitional carcinoma (EJ) cell lines.8,9,18 However, all these cell lines exhibited a range of sensitivity to the drug. When treated with MKT-077, normal cells showed none of the symptoms reported in cancerous cell lines.15 However, it caused eventual irreversible renal toxicity in clinical trials.19
Mapping the human sperm proteome – novel insights into reproductive research
Published in Expert Review of Proteomics, 2023
Mika Alexia Miyazaki, Raquel Lozano Guilharducci, Paula Intasqui, Ricardo Pimenta Bertolla
In addition, heat shock protein family A (HSP70) member 2 and member 9 (HSPA2 and HSPA9) were shown to be deregulated. In previous studies with HSPA2-null mice, there were several germ cells and spermatogenesis cells getting into apoptosis, when compared to wild-type mice, leading these animals to be infertile [59,60]. These HSP70 family of proteins participates in mitochondrial protein folding and energy generation. Once again, results converge to energy metabolism alterations, but, in this case, the altered protein levels were from post-glycolytic proteins, what confirms the crucial role of mitochondria for sperm motility [52,54].