scholarly journals Evaluation of artificial signal peptides for secretion of two lysosomal enzymes in CHO cells

2021 ◽  
Author(s):  
Kai-Wen Cheng ◽  
Feng Wang ◽  
George A Lopez ◽  
Srikanth Singamsetty ◽  
Jill Wood ◽  
...  
Keyword(s):  
Signal Peptide ◽  
Light Chain ◽  
Lysosomal Enzyme ◽  
Mammalian Cells ◽  
Lysosomal Enzymes ◽  
Chinese Hamster ◽  
Signal Peptides ◽  
Clinical Use ◽  
Enzyme Replacement ◽  
The Impact

Enzyme replacement therapy (ERT) is a scientifically rational and clinically proven treatment for lysosomal storage diseases. Most enzymes used for ERT are purified from the culture supernatant of mammalian cells. However, it is challenging to purify lysosomal enzymes with sufficient quality and quantity for clinical use due to their low secretion levels in mammalian cell systems. To improve the secretion efficiency of recombinant lysosomal enzymes, we evaluated the impact of artificial signal peptides on the production of recombinant lysosomal enzymes in Chinese Hamster Ovary (CHO) cell lines. We engineered two recombinant human lysosomal enzymes, N-acetyl-a-glucosaminidase (rhNAGLU) and glucosamine (N-acetyl)-6-sulfatase (rhGNS), by replacing their native signal peptides with 9 different signal peptides derived from highly secretory proteins and expressed them in CHO K1 cells. When comparing the native signal peptides, we found that rhGNS was secreted into media at higher levels than rhNAGLU. The secretion of rhNAGLU and rhGNS can, however, be carefully controlled by altering signal peptides. The secretion of rhNAGLU was relatively higher with murine Igk light chain and human chymotrypsinogen B1 signal peptides, whereas Igk light chain signal peptide 1 and human chymotrypsinogen B1 signal peptides were more effective for rhGNS secretion, suggesting that human chymotrypsinogen B1 signal peptide is the most appropriate for increasing lysosomal enzyme secretion.  Collectively, our results indicate that altering signal peptide can modulate the secretion of recombinant lysosome enzymes and will enable lysosomal enzyme production for clinical use.

scholarly journals Proteomics identifies signal peptide features determining the substrate specificity in human Sec62/Sec63-dependent ER protein import

2019 ◽  
Author(s):  
Stefan Schorr ◽  
Duy Nguyen ◽  
Sarah Haßdenteufel ◽  
Nagarjuna Nagaraj ◽  
Adolfo Cavalié ◽  
...  
Keyword(s):  
Signal Peptide ◽  
Mammalian Cells ◽  
Protein Import ◽  
Supplementary Information ◽  
Signal Peptides ◽  
Proteomics Data ◽  
Amino Terminal ◽  
Link Type ◽  
Sec61 Channel

AbstractIn mammalian cells one-third of all polypeptides are integrated into the membrane or translocated into the lumen of the endoplasmic reticulum (ER) via the Sec61-channel. While the Sec61-complex facilitates ER-import of most precursor polypeptides, the Sec61-associated Sec62/Sec63-complex supports ER-import in a substrate-specific manner. So far, mainly posttranslationally imported precursors and the two cotranslationally imported precursors of ERj3 and prion protein were found to depend on the Sec62/Sec63-complex in vitro. Therefore, we determined the rules for engagement of Sec62/Sec63 in ER-import in intact human cells using a recently established unbiased proteomics approach. In addition to confirming ERj3, we identified twenty-two novel Sec62/Sec63-substrates under these in vivo-like conditions. As a common feature, those previously unknown substrates share signal peptides with comparatively longer but less hydrophobic H-region and lower C-region polarity. Further analyses with four substrates, and ERj3 in particular, revealed the combination of a slowly-gating signal peptide and a downstream translocation-disruptive positively charged cluster of amino acid residues as decisive for the Sec62-/Sec63-requirement. In the case of ERj3, these features were found to be responsible for an additional BiP-requirement and to correlate with sensitivity towards the Sec61-channel inhibitor CAM741. Thus, the human Sec62/Sec63-complex may support Sec61-channel opening for precursor polypeptides with slowly-gating signal peptides by direct interaction with the cytosolic amino-terminal peptide of Sec61α or via recruitment of BiP and its interaction with the ER-lumenal loop 7 of Sec61α. These novel insights into the mechanism of human ER protein import contribute to our understanding of the etiology of SEC63-linked Polycystic Liver Disease.DatabasesThe mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository (http://www.ebi.ac.uk/pride/archive/projects/Identifiers) with the dataset identifiers: PXD008178, PXD011993, and PXD012078. Supplementary information was deposited at Mendeley Data under the DOI:10.17632/6s5hn73jcv.1 (http://dx.doi.or/10.17632/6s5hn73jcv.1).

Ultrastructural Changes in Three Different Mammalian Cells Following Ultraviolet Laser Irradiation

1972 ◽  
Vol 30 ◽  
pp. 350-351
Author(s):  
K. Shankar Narayan ◽  
Kailash C. Gupta ◽  
Tohru Okigaki
Keyword(s):  
Ultraviolet Light ◽  
Mammalian Cells ◽  
Biological Effects ◽  
Survival Rates ◽  
Chinese Hamster ◽  
Cell Types ◽  
Differential Sensitivity ◽  
Ultrastructural Changes ◽  
Ultraviolet Laser

The biological effects of short-wave ultraviolet light has generally been described in terms of changes in cell growth or survival rates and production of chromosomal aberrations. Ultrastructural changes following exposure of cells to ultraviolet light, particularly at 265 nm, have not been reported.We have developed a means of irradiating populations of cells grown in vitro to a monochromatic ultraviolet laser beam at a wavelength of 265 nm based on the method of Johnson. The cell types studies were: i) WI-38, a human diploid fibroblast; ii) CMP, a human adenocarcinoma cell line; and iii) Don C-II, a Chinese hamster fibroblast cell strain. The cells were exposed either in situ or in suspension to the ultraviolet laser (UVL) beam. Irradiated cell populations were studied either "immediately" or following growth for 1-8 days after irradiation.Differential sensitivity, as measured by survival rates were observed in the three cell types studied. Pattern of ultrastructural changes were also different in the three cell types.

scholarly journals Baicalin Represses Type Three Secretion System of Pseudomonas aeruginosa through PQS System

Molecules ◽  
2021 ◽  
Vol 26 (6) ◽  
pp. 1497
Author(s):  
Pansong Zhang ◽  
Qiao Guo ◽  
Zhihua Wei ◽  
Qin Yang ◽  
Zisheng Guo ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Virulence Factors ◽  
Mammalian Cells ◽  
Secretion System ◽  
Chinese Herbs ◽  
Infection Model ◽  
Lung Pathology ◽  
Promising Alternative ◽  
The Impact

Therapeutics that target the virulence of pathogens rather than their viability offer a promising alternative for treating infectious diseases and circumventing antibiotic resistance. In this study, we searched for anti-virulence compounds against Pseudomonas aeruginosa from Chinese herbs and investigated baicalin from Scutellariae radix as such an active anti-virulence compound. The effect of baicalin on a range of important virulence factors in P. aeruginosa was assessed using luxCDABE-based reporters and by phenotypical assays. The molecular mechanism of the virulence inhibition by baicalin was investigated using genetic approaches. The impact of baicalin on P. aeruginosa pathogenicity was evaluated by both in vitro assays and in vivo animal models. The results show that baicalin diminished a plenty of important virulence factors in P. aeruginosa, including the Type III secretion system (T3SS). Baicalin treatment reduced the cellular toxicity of P. aeruginosa on the mammalian cells and attenuated in vivo pathogenicity in a Drosophila melanogaster infection model. In a rat pulmonary infection model, baicalin significantly reduced the severity of lung pathology and accelerated lung bacterial clearance. The PqsR of the Pseudomonas quinolone signal (PQS) system was found to be required for baicalin’s impact on T3SS. These findings indicate that baicalin is a promising therapeutic candidate for treating P. aeruginosa infections.

Thermotropic lipid and protein transitions in Chinese hamster lung cell membranes: relationship to hyperthermic cell killing

1983 ◽  
Vol 61 (6) ◽  
pp. 421-427 ◽  
Author(s):  
James R. Lepock ◽  
Kwan-Hon Cheng ◽  
Hisham Al-Qysi ◽  
Jack Kruuv
Keyword(s):  
Mammalian Cells ◽  
Plasma Membranes ◽  
Chinese Hamster ◽  
Cell Killing ◽  
Water Soluble ◽  
Growth Data ◽  
Protein Fluorescence ◽  
Polar Groups ◽  
Spin Resonance ◽  
Intrinsic Protein Fluorescence

Exposure of mammalian cells to hyperthermic temperatures (ca. 41–45 °C) appears to act as a direct or triggering effect to produce some later response such as cell death, thermotolerance, or heat-shock protein synthesis. The high activation energy of cell killing indicates that the direct effect of hyperthermia might be a thermotropic transition in some cellular component, for this particular response. Both hyperthermic survival and growth data imply that the temperature for the onset of hyperthermic cell killing is 40–41.5 °C for Chinese hamster lung V79 cells. Studies using the electron spin resonance label 2,2-dimethyl-5-dodecyl-5-methyloxazolidine-N-oxide and the fluorescent probe 1,6-diphenyl-1,3,5-hexatriene show the existence of lipid transitions at approximately 7–8 and 23–36 °C (or a broad transition between these temperatures) in mitochondria and whole cell homogenates, that correlate well with changes in growth and hypothermic killing. No lipid transition was detected near 40–41.5 °C that could correlate with hyperthermic killing in either mitochondrial or plasma membranes, but measurements of intrinsic protein fluorescence and protein fluorophore to trans-paranaric acid energy transfer demonstrate the existence of an irreversible transition in protein structure or arrangement above ca. 40 °C in both mitochondrial and plasma membranes. This transition is due to protein rearrangement and (or) unfolding such that there is increased exposure of protein tryptophan and tyrosine residues to polar groups and to paranaric acid. The strength of the transition implies that a significant fraction of total membrane protein is involved in this transition, which may be analogous to the heat-induced denaturation of water-soluble proteins. This alteration in membrane structure above ca. 40 °C could cause many of the observed changes in plasma membrane and mitochondrial function, which may further be involved in cellular responses to hyperthermia.

scholarly journals m5U54 tRNA Hypomodification by Lack of TRMT2A Drives the Generation of tRNA-Derived Small RNAs

2021 ◽  
Vol 22 (6) ◽  
pp. 2941
Author(s):  
Marisa Pereira ◽  
Diana R. Ribeiro ◽  
Miguel M. Pinheiro ◽  
Margarida Ferreira ◽  
Stefanie Kellner ◽  
...  
Keyword(s):  
Stress Response ◽  
Small Rnas ◽  
Mammalian Cells ◽  
Cellular Stress ◽  
Cellular Stress Response ◽  
Translation Regulation ◽  
Translation Efficiency ◽  
Down Regulation ◽  
Gene Expression Control ◽  
The Impact

Transfer RNA (tRNA) molecules contain various post-transcriptional modifications that are crucial for tRNA stability, translation efficiency, and fidelity. Besides their canonical roles in translation, tRNAs also originate tRNA-derived small RNAs (tsRNAs), a class of small non-coding RNAs with regulatory functions ranging from translation regulation to gene expression control and cellular stress response. Recent evidence indicates that tsRNAs are also modified, however, the impact of tRNA epitranscriptome deregulation on tsRNAs generation is only now beginning to be uncovered. The 5-methyluridine (m5U) modification at position 54 of cytosolic tRNAs is one of the most common and conserved tRNA modifications among species. The tRNA methyltransferase TRMT2A catalyzes this modification, but its biological role remains mostly unexplored. Here, we show that TRMT2A knockdown in human cells induces m5U54 tRNA hypomodification and tsRNA formation. More specifically, m5U54 hypomodification is followed by overexpression of the ribonuclease angiogenin (ANG) that cleaves tRNAs near the anticodon, resulting in accumulation of 5′tRNA-derived stress-induced RNAs (5′tiRNAs), namely 5′tiRNA-GlyGCC and 5′tiRNA-GluCTC, among others. Additionally, transcriptomic analysis confirms that down-regulation of TRMT2A and consequently m5U54 hypomodification impacts the cellular stress response and RNA stability, which is often correlated with tiRNA generation. Accordingly, exposure to oxidative stress conditions induces TRMT2A down-regulation and tiRNA formation in mammalian cells. These results establish a link between tRNA hypomethylation and ANG-dependent tsRNAs formation and unravel m5U54 as a tRNA cleavage protective mark.

Intramembrane proteolysis and post-targeting functions of signal peptides

2003 ◽  
Vol 31 (6) ◽  
pp. 1243-1247 ◽  
Author(s):  
B. Martoglio
Keyword(s):  
Endoplasmic Reticulum ◽  
Lipid Bilayer ◽  
Signal Peptide ◽  
Eukaryotic Cells ◽  
Endoplasmic Reticulum Membrane ◽  
Signal Peptides ◽  
Peptide Fragments ◽  
Intramembrane Proteolysis ◽  
Signal Sequences ◽  
Membrane Spanning

Signal sequences are the addresses of proteins destined for secretion. In eukaryotic cells, they mediate targeting to the endoplasmic reticulum membrane and insertion into the translocon. Thereafter, signal sequences are cleaved from the pre-protein and liberated into the endoplasmic reticulum membrane. We have recently reported that some liberated signal peptides are further processed by the intramembrane-cleaving aspartic protease signal peptide peptidase. Cleavage in the membrane-spanning portion of the signal peptide promotes the release of signal peptide fragments from the lipid bilayer. Typical processes that include intramembrane proteolysis is the regulatory or signalling function of cleavage products. Likewise, signal peptide fragments liberated upon intramembrane cleavage may promote such post-targeting functions in the cell.

scholarly journals An interaction between the mammalian DNA repair protein XRCC1 and DNA ligase III.

1994 ◽  
Vol 14 (1) ◽  
pp. 68-76 ◽  
Author(s):  
K W Caldecott ◽  
C K McKeown ◽  
J D Tucker ◽  
S Ljungquist ◽  
L H Thompson
Keyword(s):  
Dna Repair ◽  
Affinity Chromatography ◽  
Mammalian Cells ◽  
Excision Repair ◽  
Affinity Purification ◽  
Alkylating Agents ◽  
Chinese Hamster ◽  
Dna Ligase ◽  
Base Excision ◽  
Dna Ligase Iii

XRCC1, the human gene that fully corrects the Chinese hamster ovary DNA repair mutant EM9, encodes a protein involved in the rejoining of DNA single-strand breaks that arise following treatment with alkylating agents or ionizing radiation. In this study, a cDNA minigene encoding oligohistidine-tagged XRCC1 was constructed to facilitate affinity purification of the recombinant protein. This construct, designated pcD2EHX, fully corrected the EM9 phenotype of high sister chromatid exchange, indicating that the histidine tag was not detrimental to XRCC1 activity. Affinity chromatography of extract from EM9 cells transfected with pcD2EHX resulted in the copurification of histidine-tagged XRCC1 and DNA ligase III activity. Neither XRCC1 or DNA ligase III activity was purified during affinity chromatography of extract from EM9 cells transfected with pcD2EX, a cDNA minigene that encodes untagged XRCC1, or extract from wild-type AA8 or untransfected EM9 cells. The copurification of DNA ligase III activity with histidine-tagged XRCC1 suggests that the two proteins are present in the cell as a complex. Furthermore, DNA ligase III activity was present at lower levels in EM9 cells than in AA8 cells and was returned to normal levels in EM9 cells transfected with pcD2EHX or pcD2EX. These findings indicate that XRCC1 is required for normal levels of DNA ligase III activity, and they implicate a major role for this DNA ligase in DNA base excision repair in mammalian cells.

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