scholarly journals The desmin mutation R349P increases contractility and fragility of stem cell-generated muscle micro-tissues

2021 ◽  
Author(s):  
Marina Spoerrer ◽  
Delf Kah ◽  
Richard C Gerum ◽  
Barbara Reischl ◽  
Danyil Huraskin ◽  
...  
Keyword(s):  
Striated Muscle ◽  
Contractile Function ◽  
Three Dimensional ◽  
Mechanical Damage ◽  
Underlying Mechanism ◽  
Intermediate Filament Protein ◽  
Wild Type ◽  
Age Related ◽  
Contractile Forces

Desminopathies comprise hereditary myopathies and cardiomyopathies caused by mutations in the intermediate filament protein desmin that lead to severe and often lethal degeneration of striated muscle tissue. Animal and single cell studies hinted that this degeneration process is associated with massive ultrastructural defects correlating with increased susceptibility of the muscle to acute mechanical stress. The underlying mechanism of mechanical susceptibility, and how muscle degeneration develops over time, however, has remained elusive. Here, we investigated the effect of a desmin mutation on the formation, differentiation, and contractile function of in vitro-engineered three-dimensional micro-tissues grown from muscle stem cells (satellite cells) isolated from heterozygous R349P desmin knock-in mice. Micro-tissues grown from desmin-mutated cells exhibited spontaneous unsynchronized contractions, higher contractile forces in response to electrical stimulation, and faster force recovery compared to tissues grown from wild-type cells. Within one week of culture, the majority of R349P desmin-mutated tissues disintegrated, whereas wild-type tissues remained intact over at least three weeks. Moreover, under tetanic stimulation lasting less than five seconds, desmin-mutated tissues partially or completely ruptured, whereas wild-type tissues did not display signs of damage. Our results demonstrate that the progressive degeneration of desmin-mutated micro-tissues is closely linked to extracellular matrix fiber breakage associated with increased contractile forces and unevenly distributed tensile stress. This suggests that the age-related degeneration of skeletal and cardiac muscle in patients suffering from desminopathies may be similarly exacerbated by mechanical damage from high-intensity muscle contractions. We conclude that micro-tissues may provide a valuable tool for studying the organization of myocytes and the pathogenic mechanisms of myopathies.

scholarly journals Effects of CREG1 on Age-Associated Metabolic Phenotypes and Renal Senescence in Mice

2021 ◽  
Vol 22 (3) ◽  
pp. 1276
Author(s):  
Michihiro Hashimoto ◽  
Ayumi Goto ◽  
Yuki Endo ◽  
Masataka Sugimoto ◽  
Jun Ueda ◽  
...  
Keyword(s):  
Renal Dysfunction ◽  
Body Weight Gain ◽  
Mrna Levels ◽  
Wild Type ◽  
Metabolic Phenotypes ◽  
Age Related ◽  
Secretory Phenotype ◽  
Filtering Function ◽  
Renal Senescence

Cellular repressor of E1A-stimulated genes 1 (CREG1) is a secreted glycoprotein that accelerates p16-dependent cellular senescence in vitro. We recently reported the ability of CREG1 to stimulate brown adipogenesis using adipocyte P2-CREG1-transgenic (Tg) mice; however, little is known about the effect of CREG1 on aging-associated phenotypes. In this study, we investigated the effects of CREG1 on age-related obesity and renal dysfunction in Tg mice. Increased brown fat formation was detected in aged Tg mice, in which age-associated metabolic phenotypes such as body weight gain and increases in blood glucose were improved compared with those in wild-type (WT) mice. Blood CREG1 levels increased significantly in WT mice with age, whereas the age-related increase was suppressed, and its levels were reduced, in the livers and kidneys of Tg mice relative to those in WT mice at 25 months. Intriguingly, the mRNA levels of Ink4a, Arf, and senescence-associated secretory phenotype (SASP)-related genes and p38MAPK activity were significantly lowered in the aged kidneys of Tg mice, in which the morphological abnormalities of glomeruli as well as filtering function seen in WT kidneys were alleviated. These results suggest the involvement of CREG1 in kidney aging and its potential as a target for improving age-related renal dysfunction.

scholarly journals Increased Association of Deamidated αA-N101D with Lens Membrane of Transgenic αAN101D vs. Wild Type αA Mice: Potential Effects on Intracellular Ionic Imbalance and Membrane Disorganization

2020 ◽  
Author(s):  
Om Srivast ◽  
Kiran Srivast ◽  
Roy Joseph ◽  
Landon Wilson
Keyword(s):  
Epithelial Cells ◽  
Western Blot ◽  
Immunogold Labeling ◽  
Human Lens ◽  
Membrane Association ◽  
Related Protein ◽  
Wild Type ◽  
Age Related ◽  
Ionic Imbalance

Abstract We have generated two mouse models, in one by inserting the human lens αAN101D transgene in CRYαAN101D mice, and in the other by inserting human wild-type αA-transgene in CRYαAWT mice. The CRYαAN101D mice developed cortical cataract at about 7-months of age relative to CRYαAWT mice. The objective of the study was to determine the following relative changes in the lenses of CRYαAN101D- vs. CRYαAWT mice: age-related changes with specific emphasis on protein insolubilization, relative membrane-association of αAN101D vs. WTαA proteins, and changes in intracellular ionic imbalance and membrane organization. Methods: Lenses of varying ages from CRYαAWT and CRYαAN101D mice were compared for an age-related protein insolubilization. The relative lens membrane-association of the αAN101D- and WTαA proteins in the two types of mice was determined by immunohistochemical-, immunogold-labeling-, and western blot analyses. The relative levels of membrane-binding of recombinant αAN101D- and WTαA proteins was determined by an in vitro assay, and the levels of intracellular Ca2+ uptake and Na, K-ATPase mRNA were determined in the cultured epithelial cells from lenses of the two types of mice.Results: Compared to the lenses of CRYαAWT, the lenses of CRYαAN101D mice exhibited: (A) An increase in age-related protein insolubilization beginning at about 4-months of age. (B) A greater lens membrane-association of αAN101D- relative to WTαA protein during immunogold-labeling- and western blot analyses, including relatively a greater membrane swelling in the CRYαAN101D lenses. (C) During in vitro assay, the greater levels of binding αAN101D- relative to WTαA protein to membranes was observed. (D) The 75% lower level of Na, K-ATPase mRNA but 1.5X greater Ca2+ uptake were observed in cultured lens epithelial cells of CRYαAN101D- than those of CRYαAWT mice. Conclusions: The results show that an increased lens membrane association of αAN101D--relative WTαA protein in CRYαAN101D mice than CRYαAWT mice occurs, which causes intracellular ionic imbalance, and in turn, membrane swelling that potentially leads to cortical opacity.

scholarly journals Reversible autoinhibitory regulation ofEscherichia colimetallopeptidase BepA for selective β-barrel protein degradation

2020 ◽  
Vol 117 (45) ◽  
pp. 27989-27996
Author(s):  
Yasushi Daimon ◽  
Shin-ichiro Narita ◽  
Ryoji Miyazaki ◽  
Yohei Hizukuri ◽  
Hiroyuki Mori ◽  
...  
Keyword(s):  
Active Site ◽  
Protease Activity ◽  
Underlying Mechanism ◽  
Zinc Ion ◽  
Wild Type ◽  
Protease Domain ◽  
Loop Region ◽  
Assembly Pathway

Escherichia coliperiplasmic zinc-metallopeptidase BepA normally functions by promoting maturation of LptD, a β-barrel outer-membrane protein involved in biogenesis of lipopolysaccharides, but degrades it when its membrane assembly is hampered. These processes should be properly regulated to ensure normal biogenesis of LptD. The underlying mechanism of regulation, however, remains to be elucidated. A recently solved BepA structure has revealed unique features: In particular, the active site is buried in the protease domain and conceivably inaccessible for substrate degradation. Additionally, the His-246 residue in the loop region containing helix α9 (α9/H246 loop), which has potential flexibility and covers the active site, coordinates the zinc ion as the fourth ligand to exclude a catalytic water molecule, thereby suggesting that the crystal structure of BepA represents a latent form. To examine the roles of the α9/H246 loop in the regulation of BepA activity, we constructed BepA mutants with a His-246 mutation or a deletion of the α9/H246 loop and analyzed their activities in vivo and in vitro. These mutants exhibited an elevated protease activity and, unlike the wild-type BepA, degraded LptD that is in the normal assembly pathway. In contrast, tethering of the α9/H246 loop repressed the LptD degradation, which suggests that the flexibility of this loop is important to the exhibition of protease activity. Based on these results, we propose that the α9/H246 loop undergoes a reversible structural change that enables His-246–mediated switching (histidine switch) of its protease activity, which is important for regulated degradation of stalled/misassembled LptD.

Chronic β-agonist administration affects cardiac function of adult but not old rats, independent of β-adrenoceptor density

2005 ◽  
Vol 289 (1) ◽  
pp. H344-H349 ◽  
Author(s):  
Paul Gregorevic ◽  
James G. Ryall ◽  
David R. Plant ◽  
Martin N. Sillence ◽  
Gordon S. Lynch
Keyword(s):  
Cardiac Function ◽  
Contractile Function ◽  
Heart Function ◽  
Relative Increase ◽  
Left Ventricular ◽  
Aged Rats ◽  
Adult Rats ◽  
Age Related ◽  
Heart Mass

Although β-adrenoceptor agonists have clinical merit for attenuating the age-related loss of skeletal muscle mass and strength (sarcopenia), potential cardiac-related side effects may limit their clinical application. The aim of this study was to determine whether chronic β-agonist administration impairs cardiac function in adult or aged rats. Adult (16 mo) and aged (28 mo) Fischer 344 rats were treated with fenoterol (1.4 mg·kg−1·day−1 ip) or vehicle for 4 wk. Heart function was assessed in vitro before analyses of cardiac structure and β-adrenoceptor density. Heart mass increased 17% and 25% in fenoterol-treated adult and aged rats, respectively. The increased heart mass in aged, but not adult, rats was associated with a relative increase in collagen content. Cardiac hypertrophy in adult rats was associated with an increase in left ventricular developed pressure, a marked reduction in cardiac output, and a reduction in coronary flow per unit heart mass. In contrast, negligible differences in ventricular function were observed in fenoterol-treated aged rats. The differential effect on contractile function was not associated with age-related differences in β-adrenoceptor density but, rather, an age-related increase in downregulation after treatment. Our results show that chronic β-agonist treatment impairs cardiac function to a greater extent in adult than in aged rats. These results provide important information regarding the potential effects of chronic β-agonist use on cardiac function and the future development of safe and effective treatments for sarcopenia.

scholarly journals Lithium ions display weak interaction with amyloid-beta (Aβ) peptides and have minor effects on their aggregation

2021 ◽  
Author(s):  
Elina Berntsson ◽  
Suman Paul ◽  
Faraz Vosough ◽  
Sabrina B. Sholts ◽  
Jüri Jarvet ◽  
...  
Keyword(s):  
Amyloid Fibrils ◽  
Amyloid Β ◽  
Underlying Mechanism ◽  
Aβ Peptides ◽  
Beneficial Effects ◽  
Incurable Disease ◽  
Age Related ◽  
Aβ Aggregation ◽  
In Vitro Interactions

Alzheimer’s disease (AD) is an incurable disease and the main cause of age-related dementia worldwide, despite decades of research. Treatment of AD with lithium (Li) has showed promising results, but the underlying mechanism is unclear. The pathological hallmark of AD brains is deposition of amyloid plaques, consisting mainly of amyloid-β (Aβ) peptides aggregated into amyloid fibrils. The plaques contain also metal ions of e.g. Cu, Fe, and Zn, and such ions are known to interact with Aβ peptides and modulate their aggregation and toxicity. The interactions between Aβ peptides and Li+ ions have however not been well investigated. Here, we use a range of biophysical techniques to characterize in vitro interactions between Aβ peptides and Li+ ions. We show that Li+ ions display weak and non-specific interactions with Aβ peptides, and have minor effects on Aβ aggregation. These results indicate that possible beneficial effects of Li on AD pathology are not likely caused by direct interactions between Aβ peptides and Li+ ions.

scholarly journals SUN-564 ATG7 Overexpression Results in Reduction of Hepatocellular Lipid Content in Vitro

2020 ◽  
Vol 4 (Supplement_1) ◽  
Author(s):  
Federica Tavaglione ◽  
Guido Baselli ◽  
Ester Ciociola ◽  
Umberto Vespasiani Gentilucci ◽  
Luca Valenti ◽  
...  
Keyword(s):  
Liver Disease ◽  
Lipid Content ◽  
Lipid Droplets ◽  
Low Frequency ◽  
Underlying Mechanism ◽  
Loss Of Function ◽  
Wild Type ◽  
Intracellular Lipid ◽  
Heparg Cells

Abstract Abstract: Non-alcoholic fatty liver disease (NAFLD) is currently the most common liver disease worldwide, paralleling the epidemic of obesity and type 2 diabetes. Despite the high prevalence of NAFLD, only a minority of patients progress to NASH and advanced fibrosis. The reasons for this inter-individual variability are not completely understood but can be partially accounted for by genetic risk factors (1). Although several common genetic variants associated with liver disease have been identified, there is still a proportion of NAFLD heritability that remains unknown. The rare rs143545741 C>T variant in the autophagy related 7 (ATG7) gene (P426L) has been associated with a higher risk of progressive NAFLD (2). Interestingly, ATG7 encodes a E1-like ubiquitin activating enzyme which is involved in hepatic lipophagy (3). We hypothesized that the unknown heritability of NAFLD might be partially explained by rare genetic variants, therefore not identified in the GWAS studies. Moreover, we assumed that loss-of-function variants in ATG7 might confer an increased susceptibility to NAFLD by reducing autophagic catabolism of lipid droplets in the liver. To examine the underlying mechanism of the low-frequency V471A variant and the rare T86I, L127I, Q170E, and P426L variants in ATG7, we performed in vitro experiments of HepaRG cells overexpressing the human V5-tagged ATG7. We observed a reduction in intracellular lipid content in HepaRG cells overexpressing the ATG7 wild type and the 86I mutant protein (p=0.029, n=4) but not the 127I, 170E, 426L, and 471A mutant proteins. Cells with the ATG7 127I, 170E, 426L, and 471A mutants had higher intracellular lipid content compared to cells overexpressing the wild type protein (p=0.029, n=4). Our data suggested that the low-frequency V471A variant and the rare L127I, Q170E, and P426L variants in ATG7 are loss-of-function, resulting in defective lipophagy, reduced hepatocellular lipid droplets turnover, and excessive lipid accumulation. More experiments are needed to clarify the underlying mechanism of the T86I variant. In conclusion, we highlighted a role for ATG7 in reducing hepatocellular lipid content. Furthermore, we provided evidence showing non-synonymous variants in ATG7 increase the risk of NAFLD and that these variants are loss-of-function. We speculate that ATG7 might be a new susceptibility risk genetic locus for liver disease development and progression. References: (1) Eslam et al. J Hepatol. 2018;68(2):268–279. (2) Baselli et al. The Liver Meeting 2018 - AASLD. Hepatology. October 2018. Volume 68, Issue S1. (3) Martinez-Lopez and Singh. Annu Rev Nutr. 2015;35:215–37.

scholarly journals Increased Association of Deamidated αA-N101D with Lens Membrane of Transgenic αAN101D vs. Wild Type αA Mice: Potential Effects on Intracellular Ionic Imbalance and Membrane Disorganization 

2020 ◽  
Author(s):  
Om Srivast ◽  
Kiran Srivast ◽  
Roy Joseph ◽  
Landon Wilson
Keyword(s):  
Western Blot ◽  
In Vitro Assay ◽  
Immunogold Labeling ◽  
Membrane Association ◽  
Related Protein ◽  
Wild Type ◽  
Vitro Assay ◽  
Age Related ◽  
Ionic Imbalance

Abstract We have generated two mouse models, in one by inserting the human lens αAN101D transgene in CRYαA N101D mice, and in the other by inserting human wild-type αA-transgene in CRYαA WT mice. The CRYαA N101D mice developed cortical cataract at about 7-months of age relative to CRYαA WT mice. The objective of the study was to determine the following relative changes in the lenses of CRYαA N101D - vs. CRYαA WT mice: age-related changes with specific emphasis on protein insolubilization, relative membrane-association of αA N101D vs. WTαA proteins, and changes in intracellular ionic imbalance and membrane organization. Methods: Lenses of varying ages from CRYαA WT and CRYαA N101D mice were compared for an age-related protein insolubilization. The relative lens membrane-association of the αAN101D- and WTαA proteins in the two types of mice was determined by immunohistochemical-, immunogold-labeling-, and western blot analyses. The relative levels of membrane-binding of recombinant αA N101D - and WTαA proteins was determined by an in vitro assay, and the levels of intracellular Ca 2+ uptake and Na, K-ATPase mRNA were determined in the cultured epithelial cells from lenses of the two types of mice. Results: Compared to the lenses of CRYαA WT , the lenses of CRYαA N101D mice exhibited: (A) An increase in age-related protein insolubilization beginning at about 4-months of age. (B) A greater lens membrane-association of αAN101D- relative to WTαA protein during immunogold-labeling- and western blot analyses, including relatively a greater membrane swelling in the CRYαA N101D lenses. (C) During in vitro assay, the greater levels of binding αAN101D- relative to WTαA protein to membranes was observed. (D) The 75% lower level of Na, K-ATPase mRNA but 1.5X greater Ca 2+ uptake was observed in cultured lens epithelial cells of CRYαA N101D- than those of CRYαA WT mice. Conclusions: The results show that an increased lens membrane association of αA N101D - - relative WTαA protein in CRYαA N101D mice than CRYαA WT mice occurs, which causes intracellular ionic imbalance, and in turn, membrane swelling that potentially leads to cortical opacity.

scholarly journals Host metabolism dysregulation and cell tropism identification in human airway and alveolar organoids upon SARS-CoV-2 infection

2020 ◽  
Author(s):  
Rongjuan Pei ◽  
Jianqi Feng ◽  
Yecheng Zhang ◽  
Hao Sun ◽  
Lian Li ◽  
...  
Keyword(s):  
Cell Lines ◽  
Three Dimensional ◽  
Embryonic Stem ◽  
Neutralizing Antibody ◽  
Underlying Mechanism ◽  
Alveolar Type ◽  
Physiological Conditions ◽  
Therapeutic Drugs ◽  
Distal Airway

AbstractThe coronavirus disease 2019 (COVID-19) pandemic is caused by infection with the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which is spread primary via respiratory droplets and infects the lungs. Currently widely used cell lines and animals are unable to accurately mimic human physiological conditions because of the abnormal status of cell lines (transformed or cancer cells) and species differences between animals and humans. Organoids are stem cell-derived self-organized three-dimensional culture in vitro and model the physiological conditions of natural organs. Here we showed that SARS-CoV-2 infected and extensively replicated in human embryonic stem cells (hESCs)-derived lung organoids, including airway and alveolar organoids which covered the complete infection and spread route for SARS-CoV-2 within lungs. The infected cells were ciliated, club, and alveolar type 2 (AT2) cells, which were sequentially located from the proximal to the distal airway and terminal alveoli, respectively. Additionally, RNA-seq revealed early cell response to virus infection including an unexpected downregulation of the metabolic processes, especially lipid metabolism, in addition to the well-known upregulation of immune response. Further, Remdesivir and a human neutralizing antibody potently inhibited SARS-CoV-2 replication in lung organoids. Therefore, human lung organoids can serve as a pathophysiological model to investigate the underlying mechanism of SARS-CoV-2 infection and to discover and test therapeutic drugs for COVID-19.

scholarly journals Improving 10-deacetylbaccatin III-10-β-O-acetyltransferase catalytic fitness for Taxol production

2017 ◽  
Vol 8 (1) ◽  
Author(s):  
Bing-Juan Li ◽  
Hao Wang ◽  
Ting Gong ◽  
Jing-Jing Chen ◽  
Tian-Jiao Chen ◽  
...  
Keyword(s):  
Anticancer Drug ◽  
Active Site ◽  
Three Dimensional ◽  
Catalytic Efficiency ◽  
Dimensional Structure ◽  
Wild Type ◽  
One Pot ◽  
Natural Concentration ◽  
Taxol Production

Abstract The natural concentration of the anticancer drug Taxol is about 0.02% in yew trees, whereas that of its analogue 7-β-xylosyl-10-deacetyltaxol is up to 0.5%. While this compound is not an intermediate in Taxol biosynthetic route, it can be converted into Taxol by de-glycosylation and acetylation. Here, we improve the catalytic efficiency of 10-deacetylbaccatin III-10-O-acetyltransferase (DBAT) of Taxus towards 10-deacetyltaxol, a de-glycosylated derivative of 7-β-xylosyl-10-deacetyltaxol to generate Taxol using mutagenesis. We generate a three-dimensional structure of DBAT and identify its active site using alanine scanning and design a double DBAT mutant (DBATG38R/F301V) with a catalytic efficiency approximately six times higher than that of the wild-type. We combine this mutant with a β-xylosidase to obtain an in vitro one-pot conversion of 7-β-xylosyl-10-deacetyltaxol to Taxol yielding 0.64 mg ml−1 Taxol in 50 ml at 15 h. This approach represents a promising environmentally friendly alternative for Taxol production from an abundant analogue.

scholarly journals Role of Simian Virus 40 Vp1 Cysteines in Virion Infectivity

2000 ◽  
Vol 74 (23) ◽  
pp. 11388-11393 ◽  
Author(s):  
Peggy P. Li ◽  
Akira Nakanishi ◽  
Mary A. Tran ◽  
Adler M. Salazar ◽  
Robert C. Liddington ◽  
...  
Keyword(s):  
Life Cycle ◽  
Disulfide Bonds ◽  
Three Dimensional ◽  
Simian Virus 40 ◽  
Experimental System ◽  
Simian Virus ◽  
Dimensional Structure ◽  
Wild Type

ABSTRACT We have developed a new nonoverlapping infectious viral genome (NO-SV40) in order to facilitate structure-based analysis of the simian virus 40 (SV40) life cycle. We first tested the role of cysteine residues in the formation of infectious virions by individually mutating the seven cysteines in the major capsid protein, Vp1. All seven cysteine mutants—C9A, C49A, C87A, C104A, C207S, C254A, and C267L—retained viability. In the crystal structure of SV40, disulfide bridges are formed between certain Cys104 residues on neighboring pentamers. However, our results show that none of these disulfide bonds are required for virion infectivity in culture. We also introduced five different mutations into Cys254, the most strictly conserved cysteine across the polyomavirus family. We found that C254L, C254S, C254G, C254Q, and C254R mutants all showed greatly reduced (around 100,000-fold) plaque-forming ability. These mutants had no apparent defect in viral DNA replication. Mutant Vp1's, as well as wild-type Vp2/3, were mostly localized in the nucleus. Further analysis of the C254L mutant revealed that the mutant Vp1 was able to form pentamers in vitro. DNase I-resistant virion-like particles were present in NO-SV40-C254L-transfected cell lysate, but at about 1/18 the amount in wild-type-transfected lysate. An examination of the three-dimensional structure reveals that Cys254 is buried near the surface of Vp1, so that it cannot form disulfide bonds, and is not involved in intrapentamer interactions, consistent with the normal pentamer formation by the C254L mutant. It is, however, located at a critical junction between three pentamers, on a conserved loop (G2H) that packs against the dual interpentamer Ca2+-binding sites and the invading C-terminal helix of an adjacent pentamer. The substitution by the larger side chains is predicted to cause a localized shift in the G2H loop, which may disrupt Ca2+ ion coordination and the packing of the invading helix, consistent with the defect in virion assembly. Our experimental system thus allows dissection of structure-function relationships during the distinct steps of the SV40 life cycle.

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