Mitochondria have specialized ribosomes (mitoribosomes) dedicated to the expression of the genetic information encoded by their genomes. Here, using electron cryomicroscopy, we have determined the ...structure of the 75-component yeast mitoribosome to an overall resolution of 3.3 angstroms. The mitoribosomal small subunit has been built de novo and includes 15S ribosomal RNA (rRNA) and 34 proteins, including 14 without homologs in the evolutionarily related bacterial ribosome. Yeast-specific rRNA and protein elements, including the acquisition of a putatively active enzyme, give the mitoribosome a distinct architecture compared to the mammalian mitoribosome. At an expanded messenger RNA channel exit, there is a binding platform for translational activators that regulate translation in yeast but not mammalian mitochondria. The structure provides insights into the evolution and species-specific specialization of mitochondrial translation.
The human mitochondrial ribosome (mitoribosome) and associated proteins regulate the synthesis of 13 essential subunits of the oxidative phosphorylation complexes. We report the discovery of a ...mitoribosome-associated quality control pathway that responds to interruptions during elongation, and we present structures at 3.1- to 3.3-angstrom resolution of mitoribosomal large subunits trapped during ribosome rescue. Release factor homolog C12orf65 (mtRF-R) and RNA binding protein C6orf203 (MTRES1) eject the nascent chain and peptidyl transfer RNA (tRNA), respectively, from stalled ribosomes. Recruitment of mitoribosome biogenesis factors to these quality control intermediates suggests additional roles for these factors during mitoribosome rescue. We also report related cryo-electron microscopy structures (3.7 to 4.4 angstrom resolution) of elongating mitoribosomes bound to tRNAs, nascent polypeptides, the guanosine triphosphatase elongation factors mtEF-Tu and mtEF-G1, and the Oxa1L translocase.
Ribosome assembly is an essential and conserved process that is regulated at each step by specific factors. Using cryo-electron microscopy (cryo-EM), we visualize the formation of the conserved ...peptidyl transferase center (PTC) of the human mitochondrial ribosome. The conserved GTPase GTPBP7 regulates the correct folding of 16S ribosomal RNA (rRNA) helices and ensures 2'-O-methylation of the PTC base U3039. GTPBP7 binds the RNA methyltransferase NSUN4 and MTERF4, which sequester H68-71 of the 16S rRNA and allow biogenesis factors to access the maturing PTC. Mutations that disrupt binding of their
orthologs to the large subunit potently activate mitochondrial stress and cause viability, development, and sterility defects. Next-generation RNA sequencing reveals widespread gene expression changes in these mutant animals that are indicative of mitochondrial stress response activation. We also answer the long-standing question of why NSUN4, but not its enzymatic activity, is indispensable for mitochondrial protein synthesis.
Mitochondrial ribosomes (mitoribosomes) perform protein synthesis inside mitochondria, the organelles responsible for energy conversion and adenosine triphosphate (ATP) production in eukaryotic ...cells. To investigate their functions and structures, large-scale purification of intact mitoribosomes from mitochondria-rich animal tissues or HEK cells have been developed. However, the fast purification of mitoribosomes anchored to the mitochondrial inner membrane in complex with the Oxa1L translocase remains particularly challenging. Herein, we present a protocol recently developed and modified in our lab that provides details for the efficient isolation of intact mitoribosomes with its translocase Oxa1L. We combined the cell culture of PDE12
or wild-type HEK293 cell lines with the isolation of mitochondria and the purification steps used for the biochemical and structural studies of mitoribosomes and Oxa1L. Graphic abstract:
The protocol described herein includes two main sections: 1) isolation of mitochondria from HEK cells; and 2) purification of mitoribosome-Oxa1L from mitochondria. RB: Resuspension Buffer (see Recipes) (Created with BioRender.com).
Objectives. SSc is characterized by the overproduction of extracellular matrix (ECM) proteins, such as collagen and fibronectin, by activated fibroblasts, as well as oxidative stress. This study ...investigates the anti-fibrotic potential of the antioxidant epigallocatechin-3-gallate (EGCG) on activated dermal fibroblasts from SSc patients. Methods. Dermal fibroblasts from a cell line (AG), healthy individuals (CON) and SSc patients were treated with EGCG, TGF-β, PDGF-BB or other antioxidants antioxidants superoxide dismutase (SOD), catalase, N-acetyl-l-cysteine (NAC) and diphenyleneiodonium (DPI). Collagen type I, fibronectin, connective tissue growth factor (CTGF), α-smooth muscle actin and mitogen-activated protein (MAP) kinases were measured by ELISA and western blot. Fibroblast contractile forces were measured by collagen gel contraction. Reactive oxygen species (ROS) were assessed by dichlorofluorescein assay and nuclear factor κ beta (NF-κB) activity by DNA binding assay. Results. EGCG (1–100 µM) dose-dependently decreased collagen type I secretion in culture medium after 24 h in AG fibroblasts. Collagen type I protein expression in cell lysates was also significantly reduced by 40% in EGCG-treated cells (40 µM). Furthermore, EGCG also down-regulated TGF-β-induced collagen type I, fibronectin and CTGF. Similarly, in CON fibroblasts EGCG decreased basal and stimulated collagen type I, fibronectin and CTGF after 24 h, while in SSc the effects of the antioxidant were apparent after 48 h. Fibroblast-mediated contraction of collagen gels was inhibited by EGCG as early as 1 h in AG fibroblasts, and in the CON and SSc fibroblasts. Additionally, EGCG also inhibited TGF-β-stimulated gel contraction similar to other antioxidants DPI and NAC, but not SOD or catalase. EGCG suppressed TGF-β-induced ROS production in all fibroblasts. Furthermore, EGCG inhibited TGF-β or PDGF-BB-induced phospho-extracellular signal-regulated kinase (ERK)1/2 MAP kinase and NF-κB activity in SSc fibroblasts. Conclusion. The results suggest that the antioxidant, EGCG, can reduce ECM production, the fibrotic marker CTGF and inhibit contraction of dermal fibroblasts from SSc patients. Furthermore, EGCG was able to suppress intracellular ROS, ERK1/2 kinase signalling and NF-κB activity. Taken together, EGCG may be a possible candidate for therapeutic treatment aimed at reducing both oxidant stress and the fibrotic effects associated with SSc.
Purpose
Despite growing evidence supporting the benefits of yoga for cancer-related symptom management, yoga usage among cancer survivors is low. To translate the evidence of yoga benefits into ...community practice, it is critical to understand interest in yoga as well as barriers and preferences that influence yoga usage among cancer survivors.
Method
We conducted a cross-sectional survey study among cancer survivors, 18 years or older, with a primary diagnosis of cancer, and receiving treatment or follow-up care at outpatient clinics at five regional academic cancer center sites. We collected data and performed bivariate and multivariable analyses on self-reported yoga usage and interest in and barriers to practicing yoga, as well as preferred location and time for yoga practice.
Results
Of 857 participants, 70.0% had never practiced yoga and 52.3% were interested in practicing yoga. Among those interested, 52.5% had never practiced yoga. Lower interest was independently associated with being male (odds ratio OR = 0.30, 95% confidence interval CI = 0.20–0.44,
p
< 0.001), unemployed (OR = 0.60, 95% CI = 0.39–0.91,
p
= 0.016), and white (OR = 0.42, 95% CI = 0.23–0.78,
p
= 0.005). Commonly cited barriers among those who were interested but had never practiced were not aware of yoga benefits (36.3%), difficulty motivating (28.7%), experiencing symptoms (22.9%), and not enough time (22.0%). Participants indicated “on-site and at a studio near home” (41.5%) as preferred location and evenings (3–8 pm, 34.0%) as preferred time for yoga practice.
Conclusion
Although more than 50% of patients indicated interest in practicing yoga, use of yoga is low among cancer survivors. Barriers and patient preferences for yoga practice need to be addressed to design effective yoga programs for this population.
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