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1.
Nature ; 612(7941): 725-731, 2022 12.
Article in English | MEDLINE | ID: mdl-36517592

ABSTRACT

Ribosomes are highly sophisticated translation machines that have been demonstrated to be heterogeneous in the regulation of protein synthesis1,2. Male germ cell development involves complex translational regulation during sperm formation3. However, it remains unclear whether translation during sperm formation is performed by a specific ribosome. Here we report a ribosome with a specialized nascent polypeptide exit tunnel, RibosomeST, that is assembled with the male germ-cell-specific protein RPL39L, the paralogue of core ribosome (RibosomeCore) protein RPL39. Deletion of RibosomeST in mice causes defective sperm formation, resulting in substantially reduced fertility. Our comparison of single-particle cryo-electron microscopy structures of ribosomes from mouse kidneys and testes indicates that RibosomeST features a ribosomal polypeptide exit tunnel of distinct size and charge states compared with RibosomeCore. RibosomeST predominantly cotranslationally regulates the folding of a subset of male germ-cell-specific proteins that are essential for the formation of sperm. Moreover, we found that specialized functions of RibosomeST were not replaceable by RibosomeCore. Taken together, identification of this sperm-specific ribosome should greatly expand our understanding of ribosome function and tissue-specific regulation of protein expression pattern in mammals.


Subject(s)
Fertility , Ribosomes , Spermatozoa , Animals , Male , Mice , Cryoelectron Microscopy/methods , Peptides/chemistry , Peptides/metabolism , Protein Biosynthesis , Protein Folding , Ribosomes/metabolism , Spermatozoa/cytology , Spermatozoa/metabolism , Fertility/physiology , Organ Specificity , Ribosomal Proteins , Kidney/cytology , Testis/cytology
2.
EMBO Rep ; 23(2): e53081, 2022 02 03.
Article in English | MEDLINE | ID: mdl-34866316

ABSTRACT

Mouse embryonic stem cells (mESCs) can self-renew indefinitely and maintain pluripotency. Inhibition of mechanistic target of rapamycin (mTOR) by the kinase inhibitor INK128 is known to induce paused pluripotency in mESCs cultured with traditional serum/LIF medium (SL), but the underlying mechanisms remain unclear. In this study, we demonstrate that mTOR complex 1 (mTORC1) but not complex 2 (mTORC2) mediates mTOR inhibition-induced paused pluripotency in cells grown in both SL and 2iL medium (GSK3 and MEK inhibitors and LIF). We also show that mTORC1 regulates self-renewal in both conditions mainly through eIF4F-mediated translation initiation that targets mRNAs of both cytosolic and mitochondrial ribosome subunits. Moreover, inhibition of mitochondrial translation is sufficient to induce paused pluripotency. Interestingly, eIF4F also regulates maintenance of pluripotency in an mTORC1-independent but MEK/ERK-dependent manner in SL, indicating that translation of pluripotency genes is controlled differently in SL and 2iL. Our study reveals a detailed picture of how mTOR governs self-renewal in mESCs and uncovers a context-dependent function of eIF4F in pluripotency regulation.


Subject(s)
Eukaryotic Initiation Factor-4F , Mechanistic Target of Rapamycin Complex 1 , Mouse Embryonic Stem Cells/cytology , Pluripotent Stem Cells/cytology , Animals , Eukaryotic Initiation Factor-4F/genetics , Mechanistic Target of Rapamycin Complex 1/genetics , Mechanistic Target of Rapamycin Complex 2 , Mice
3.
Nucleic Acids Res ; 47(2): 883-898, 2019 01 25.
Article in English | MEDLINE | ID: mdl-30508117

ABSTRACT

Modified nucleosides on tRNA are critical for decoding processes and protein translation. tRNAs can be modified through 1-methylguanosine (m1G) on position 37; a function mediated by Trm5 homologs. We show that AtTRM5a (At3g56120) is a Trm5 ortholog in Arabidopsis thaliana. AtTrm5a is localized to the nucleus and its function for m1G and m1I methylation was confirmed by mutant analysis, yeast complementation, m1G nucleoside level on single tRNA, and tRNA in vitro methylation. Arabidopsis attrm5a mutants were dwarfed and had short filaments, which led to reduced seed setting. Proteomics data indicated differences in the abundance of proteins involved in photosynthesis, ribosome biogenesis, oxidative phosphorylation and calcium signalling. Levels of phytohormone auxin and jasmonate were reduced in attrm5a mutant, as well as expression levels of genes involved in flowering, shoot apex cell fate determination, and hormone synthesis and signalling. Taken together, loss-of-function of AtTrm5a impaired m1G and m1I methylation and led to aberrant protein translation, disturbed hormone homeostasis and developmental defects in Arabidopsis plants.


Subject(s)
Arabidopsis Proteins/metabolism , Arabidopsis/enzymology , Guanosine/analogs & derivatives , Inosine/analogs & derivatives , RNA, Transfer/metabolism , tRNA Methyltransferases/metabolism , Arabidopsis/growth & development , Arabidopsis Proteins/genetics , Arabidopsis Proteins/physiology , Biocatalysis , Calcium Signaling , Cell Nucleus/enzymology , Guanosine/metabolism , Indoleacetic Acids/metabolism , Inosine/metabolism , Mutation , Photosynthesis , RNA, Transfer/chemistry , Ribosomes/metabolism , tRNA Methyltransferases/genetics , tRNA Methyltransferases/physiology
4.
Nucleic Acids Res ; 43(21): 10525-33, 2015 Dec 02.
Article in English | MEDLINE | ID: mdl-26432831

ABSTRACT

During translation, elongation factor G (EF-G) plays a catalytic role in tRNA translocation and a facilitative role in ribosome recycling. By stabilizing the rotated ribosome and interacting with ribosome recycling factor (RRF), EF-G was hypothesized to induce the domain rotations of RRF, which subsequently performs the function of splitting the major intersubunit bridges and thus separates the ribosome into subunits for recycling. Here, with systematic mutagenesis, FRET analysis and cryo-EM single particle approach, we analyzed the interplay between EF-G/RRF and post termination complex (PoTC). Our data reveal that the two conserved loops (loop I and II) at the tip region of EF-G domain IV possess distinct roles in tRNA translocation and ribosome recycling. Specifically, loop II might be directly involved in disrupting the main intersubunit bridge B2a between helix 44 (h44 from the 30S subunit) and helix 69 (H69 from the 50S subunit) in PoTC. Therefore, our data suggest a new ribosome recycling mechanism which requires an active involvement of EF-G. In addition to supporting RRF, EF-G plays an enzymatic role in destabilizing B2a via its loop II.


Subject(s)
Peptide Elongation Factor G/chemistry , Protein Biosynthesis , Ribosomes/chemistry , Cryoelectron Microscopy , Mutation , Peptide Elongation Factor G/genetics , Peptide Elongation Factor G/metabolism , Protein Structure, Tertiary , RNA, Messenger/metabolism , RNA, Transfer/metabolism , Ribosomal Proteins/metabolism , Ribosomes/metabolism
5.
BMC Microbiol ; 15: 57, 2015 Mar 03.
Article in English | MEDLINE | ID: mdl-25887810

ABSTRACT

BACKGROUND: The infections caused by antibiotic multidrug-resistant bacteria seriously threaten human health. To prevent and cure the infections caused by multidrug-resistant bacteria, new antimicrobial agents are required. Antimicrobial peptides are ideal therapy candidates for antibiotic-resistant pathogens. However, due to high production costs, novel methods of large-scale production are urgently needed. RESULTS: The novel plectasin-derived antimicrobial peptide-MP1102 gene was constitutively expressed under the control of the GAP promoter. The optimum carbon source and concentration were determined, and 4% glucose (w/v) was initially selected as the best carbon source. Six media were assayed for the improved yield of recombinant MP1102 (rMP1102). The total protein and rMP1102 yield was 100.06 mg/l and 42.83 mg/l, which was accomplished via the use of medium number 1. The peptone and yeast extract from Hongrun Baoshun (HRBS, crude industrial grade, Beijing, China) more effectively improved the total protein and the yield of rMP1102 to 280.41 mg/l and 120.57 mg/l compared to 190.26 mg/l and 78.01 mg/l that resulted from Oxoid (used in the research). Furthermore, we observed that the total protein, antimicrobial activity and rMP1102 yield from the fermentation supernatant increased from 807.42 mg/l, 384,000 AU/ml, and 367.59 mg/l, respectively, in pH5.0 to 1213.64 mg/l, 153,600 AU/ml and 538.17 mg/ml, respectively in pH 6.5 in a 5-l fermenter. Accordingly, the productivity increased from 104464 AU/mg rMP1102 in pH 5.0 to a maximum of 285412 AU/mg rMP1102 in pH 6.5. Finally, the recombinant MP1102 was purified with a cation-exchange column with a yield of 376.89 mg/l, 96.8% purity, and a molecular weight of 4382.9 Da, which was consistent with its theoretical value of 4383 Da. CONCLUSIONS: It's the highest level of antimicrobial peptides expressed in Pichia pastoris using GAP promoter so far. These results provide an economical method for the high-level production of rMP1102 under the control of the GAP promoter.


Subject(s)
Antimicrobial Cationic Peptides/biosynthesis , Gene Expression , Pichia/genetics , Promoter Regions, Genetic , Recombinant Proteins/biosynthesis , Antimicrobial Cationic Peptides/genetics , Carbon/metabolism , China , Culture Media/chemistry , Molecular Sequence Data , Peptides/genetics , Pichia/growth & development , Pichia/metabolism , Recombinant Proteins/genetics , Sequence Analysis, DNA
6.
Appl Microbiol Biotechnol ; 99(6): 2649-62, 2015 Mar.
Article in English | MEDLINE | ID: mdl-25261129

ABSTRACT

A novel antimicrobial peptide MP1106 was designed based on the parental peptide plectasin with four mutational sites and a high level of expression in Pichia pastoris X-33 via the pPICZαA plasmid was achieved. The concentration of total secreted protein in the fermented supernatant was 2.134 g/l (29 °C), and the concentration of recombinant MP1106 (rMP1106) reached 1,808 mg/l after a 120-h induction in a 5-l fermentor. The rMP1106 was purified using a cation-exchange column, and the yield was 831 mg/l with 94.68 % purity. The sample exhibited a narrow spectrum against some Gram-positive bacteria and strong antimicrobial activity against Staphylococcus aureus at low minimal inhibitory concentrations (MICs) of 0.014, 1.8, 0.45, and 0.91 µM to S. aureus strains ATCC 25923, 29213, 6538, and 43300, respectively. Meanwhile, rMP1106 showed potent activity (0.03-1.8 µM) against 20 clinical isolates of methicillin-resistant S. aureus (MRSA). In addition, rMP1106 exhibited a broad range of thermostability from 20 to 100 °C. The higher antimicrobial activity of rMP1106 was maintained in neutral and alkaline environments (pH 6, 8, and 10), and its activity was slightly reduced in acidic environments (pH 2 and 4). The rMP1106 was resistant to the digestion of pepsin, snailase, and proteinase K and was sensitive to trypsin. It exhibited hemolytic activity of only 1.16 % at a concentration of 512 µg/ml and remained stable in human serum at 37 °C for 24 h. Furthermore, the activity of rMP1106 was minorly affected by 10 mM dithiothreitol and 20 % dimethylsulfoxide. Our results indicate that MP1106 can be produced on a large scale and has potential as a therapeutic drug against S. aureus.


Subject(s)
Anti-Bacterial Agents/pharmacology , Methicillin-Resistant Staphylococcus aureus/drug effects , Peptides/pharmacology , Protein Engineering , Amino Acid Sequence , Base Sequence , Erythrocytes/drug effects , Gram-Positive Bacteria , Hemolysis , Humans , Hydrogen-Ion Concentration , Microbial Sensitivity Tests , Molecular Sequence Data , Peptides/genetics , Pichia/metabolism , Protein Conformation
7.
Appl Microbiol Biotechnol ; 99(15): 6255-66, 2015 Aug.
Article in English | MEDLINE | ID: mdl-25620367

ABSTRACT

Currently, more antimicrobial drug candidates are urgently needed to combat the rise in drug-resistance among pathogenic microbes. A new antimicrobial peptide, MP1102, a variant of NZ2114, was designed, evaluated, and overexpressed in Pichia pastoris. The total secreted protein in cultures reached 695 mg/l, and the concentration of the recombinant MP1102 (rMP1102) was 292 mg/l. rMP1102 was purified from the fermentation supernatant by one-step cation exchange chromatography to obtain a yield of 197.1 mg/l with 96.4 % purity. rMP1102 exhibited potent activity against Gram-positive bacteria, and its minimum inhibitory concentrations (MICs) for four Staphyloccocus aureus (S. aureus) strains ranged from 0.028 to 0.11 µM, and it had stronger activity (MIC = 0.04 to 0.23 µM) to 20 clinical isolates of MRSA (cMRSA) than rNZ2114 (MIC = 0.11 to 0.90 µM). rMP1102 was shown to kill over 99.9 % of tested S. aureus cells within 6 h when treated at one, two, and four times its MIC and over 90 % of S. aureus cells within 12 h at concentrations of 5, 10, and 20 mg/kg in a mouse thigh infection model. The higher sensitivity of MRSA to MP1102 than to its parental peptide, NZ2114, indicated by this initial pharmacodynamic analysis suggests a possible difference in the killing mechanism of these two molecules. rMP1102 caused less than 0.05 % hemolytic activity at 128 µg/ml and exhibited good thermostability from 20 to 80 °C, with its highest activity being observed at pH 8.0. These results suggest that this yeast expression system is feasible for large-scale production, and rMP1102 exerted stronger activity against S. aureus than NZ2114 via a different mechanism and exhibited potential as a new antimicrobial agent for S. aureus, especially MRSA infections.


Subject(s)
Antimicrobial Cationic Peptides/pharmacology , Methicillin-Resistant Staphylococcus aureus/drug effects , Staphylococcal Infections/microbiology , Animals , Antimicrobial Cationic Peptides/genetics , Antimicrobial Cationic Peptides/isolation & purification , Chromatography, Ion Exchange , Disease Models, Animal , Gene Expression , Methicillin-Resistant Staphylococcus aureus/physiology , Mice , Microbial Sensitivity Tests , Microbial Viability/drug effects , Pichia/genetics , Pichia/metabolism , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Recombinant Proteins/pharmacology
8.
ACS Appl Mater Interfaces ; 12(7): 7931-7941, 2020 Feb 19.
Article in English | MEDLINE | ID: mdl-32003218

ABSTRACT

Multicellular spheroids can mimic the in vivo microenvironment and maintain the unique functions of tissues, which has attracted great attention in tissue engineering. However, the traditional culture microenvironment with structural deficiencies complicates the culture and collection process and tends to lose the function of multicellular spheroids with the increase of cell passage. In order to construct efficient and functional multicellular spheroids, in this study, a chitosan/polyvinyl alcohol nanofiber sponge which has an open-cell cellular structure is obtained. The hair follicle (HF) regeneration model was employed to evaluate HF-inducing ability of dermal papilla (DP) multicellular spheroids which formed on the cellular structure nanofiber sponge. Through structural fine-tuning, the nanofiber sponge has appropriate elasticity for the creation of a three-dimensional dynamic microenvironment to regulate cellular behavior. The cellular structure nanofiber sponge tilts the balance of cell-substratum and cell-cell interactions to a state which is more conducive to the formation of controllable multicellular spheroids in a short time. More importantly, it improves the secretory activity of high-passaged dermal papilla cells and restores their intrinsic properties. Experiments using BALB/c nude mice show that cultured DP multicellular spheroids could effectively enhance HF-inducing ability. This novel system provides a simple and efficient strategy for multicellular spheroid formation and HF regeneration.


Subject(s)
Dermis/physiology , Hair Follicle/physiology , Nanofibers/chemistry , Regeneration/physiology , Tissue Engineering/methods , Animals , Cell Adhesion/physiology , Cell Culture Techniques , Cell Survival/physiology , Cellular Microenvironment/physiology , Chitosan/chemistry , Dermis/cytology , Dermis/metabolism , Epithelial Cells/cytology , Epithelial Cells/metabolism , Epithelial Cells/physiology , Hair Follicle/metabolism , Mass Spectrometry , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Nude , Microscopy, Electron, Scanning , Nanofibers/ultrastructure , Polyvinyl Alcohol/chemistry , Spheroids, Cellular/metabolism , Spheroids, Cellular/physiology , Tissue Scaffolds/chemistry
9.
Int J Biol Macromol ; 160: 1212-1219, 2020 Oct 01.
Article in English | MEDLINE | ID: mdl-32485248

ABSTRACT

Ribosome recycling is the final step of the cyclic process of translation, where the post-termination complex (PoTC) is disassembled by the concerted action of ribosome recycling factor (RRF) and elongation factor G (EF-G) in the sub-second time range. Since, however, both the RRF and PoTC display highly dynamic action during this process, it is difficult to assess the molecular details of the interactions between the factors and the ribosome that are essential for rapid subunit separation. Here we characterized the molecular dynamics of RRF and PoTC by combined use of molecular dynamics simulations, single molecule fluorescence detection and single-particle cryo-EM analysis, with time resolutions in the sub-millisecond to minute range. We found that RRF displays two-layer dynamics: intra- and inter-molecular dynamics during ribosome splitting. The intra-molecular dynamics exhibits two different configurations of RRF: 'bent' and 'extended'. A single-site mutant of RRF increases its propensity to the 'extended' conformation and leads to a higher binding affinity of RRF to the PoTC. The inter-molecular dynamics between RRF and EF-G in the PoTC reveals that the domain IV of EF-G pushes against the domain II of RRF, triggering the disruption of the major inter-subunit bridge B2a, and catalyzes the splitting.


Subject(s)
Escherichia coli Proteins/chemistry , Molecular Dynamics Simulation , Ribosomal Proteins/chemistry , Ribosomes/chemistry , Escherichia coli Proteins/metabolism , Peptide Chain Termination, Translational , Ribosomal Proteins/metabolism , Ribosomes/metabolism
10.
Biomaterials ; 204: 70-79, 2019 06.
Article in English | MEDLINE | ID: mdl-30901728

ABSTRACT

Effective bleeding control and wound healing are very important and can be life saving. However, traditional wound dressings with structural deficiencies are not effective in controlling bleeding and promoting the regeneration of functional tissues. In this study, a three-dimensional (3D) layered nanofiber sponge was obtained by expanding two-dimensional (2D) nanofiber membranes into the third dimension. This sponge has a layered nanofiber structure, which increases the interfacial interaction between the sponge and blood cells to accelerate hemostasis. Through fine-tuning of structure, the 3D nanofiber sponge acquires properties beneficial to wound healing such as good elasticity and high permeability and fluid absorption ratio. The 3D nanofiber sponges are both highly compressible and resilient, providing tamponade for deep wounds and creating a good 3D dynamic microenvironment to regulate cellular behavior. Further research has demonstrated that the layered nanofiber structure could promote the regeneration of functional dermis and the restoration of differentiated adipocytes during the early repair phase. Experiments using model mice with full-thickness skin defects have shown that the layered nanofiber structure could effectively accelerate wound healing and reduce scar formation. This layered 3D nanofiber sponge design is easy to produce. Due to its excellent wound healing property, this porous nanofiber sponge has great potential for future clinical application as wound dressings.


Subject(s)
Blood Coagulation/physiology , Nanofibers/chemistry , Wound Healing , 3T3 Cells , Adipocytes/cytology , Animals , Cell Adhesion , Cell Proliferation , Chitosan/chemistry , Compressive Strength , Dermis/physiology , Elasticity , Mice , Mice, Inbred C57BL , Nanofibers/ultrastructure , Neovascularization, Physiologic , Polyvinyl Alcohol/chemistry , Regeneration , Tensile Strength
11.
ACS Appl Mater Interfaces ; 10(26): 22767-22775, 2018 Jul 05.
Article in English | MEDLINE | ID: mdl-29897733

ABSTRACT

Gene interference-based therapeutics represent a fascinating challenge and show enormous potential for cancer treatment, in which microRNA is used to correct abnormal gene. On the basis of the above, we introduced microRNA-31 to bind to 3'-untranslated region of mtEF4, resulting in the downregulation of its messenger RNA and protein to trigger cancer cells apoptosis through mitochondria-related pathway. To achieve better therapeutic effect, a mesoporous silica nanoparticle-based controlled nanoplatform had been developed. This system was fabricated by conjugation of microRNA-31 onto doxorubicin-loaded mesoporous silica nanoparticles with a poly(ethyleneimine)/hyaluronic acid coating, and drug release was triggered by acidic environment of tumors. By feat of surface functionalization and tumor-specific conjugation to nanoparticles, our drug delivery system could promote intracellular accumulation of drugs via the active transport at tumor site. More importantly, microRNA-31 not only directly targeted to mtEF4 to promote cell's death, but had synergistic effects when used in combination with doxorubicin, and achieved excellent superadditive effects. As such, our research might provide new insights toward detecting high mtEF4 cancer and exploiting highly effective anticancer drugs.


Subject(s)
Nanoparticles , Doxorubicin , Drug Delivery Systems , Humans , MicroRNAs , Neoplasms , Porosity , Silicon Dioxide
12.
Free Radic Biol Med ; 100: 231-237, 2016 11.
Article in English | MEDLINE | ID: mdl-27101739

ABSTRACT

Mitochondria are semi-autonomous organelle possessing their own translation machinery to biosynthesize mitochondrial DNA (mtDNA)-encoded polypeptides, which are the core subunits of oxidative phosphorylation (OXPHOS) complexes. Mitochondrial translation elongation factor 4 (mtEF4) is a key quality control factor in mitochondrial translation (mt-translation) that regulates mitochondrial tRNA translocation and modulates cellular responses by influencing cytoplasmic translation (ct-translation). In addition to mtEF4, mt-translational activators, mitochondrial microRNAs (mitomiRs), and MITRAC have been reported recently as crucial mt-translation regulators. Here, we focus on the novel ways how these factors regulate mt-translation, discuss the main cellular response of mammalian target of rapamycin (mTOR) signalling upon mt-translation defects, and summarize the related human diseases.


Subject(s)
Mitochondria/metabolism , Signal Transduction , TOR Serine-Threonine Kinases/metabolism , Gene Expression Regulation , Humans , Mitochondria/genetics , Protein Biosynthesis
13.
Nat Struct Mol Biol ; 23(5): 441-9, 2016 05.
Article in English | MEDLINE | ID: mdl-27065197

ABSTRACT

Elongation factor 4 (EF4) is a key quality-control factor in translation. Despite its high conservation throughout evolution, EF4 deletion in various organisms has not yielded a distinct phenotype. Here we report that genetic ablation of mitochondrial EF4 (mtEF4) in mice causes testis-specific dysfunction in oxidative phosphorylation, leading to male infertility. Deletion of mtEF4 accelerated mitochondrial translation at the cost of producing unstable proteins. Somatic tissues overcame this defect by activating mechanistic (mammalian) target of rapamycin (mTOR), thereby increasing rates of cytoplasmic translation to match rates of mitochondrial translation. However, in spermatogenic cells, the mTOR pathway was downregulated as part of the developmental program, and the resulting inability to compensate for accelerated mitochondrial translation caused cell-cycle arrest and apoptosis. We detected the same phenotype and molecular defects in germline-specific mtEF4-knockout mice. Thus, our study demonstrates cross-talk between mtEF4-dependent quality control in mitochondria and cytoplasmic mTOR signaling.


Subject(s)
Mitochondria/enzymology , Peptide Initiation Factors/physiology , Protein Biosynthesis , Spermatogenesis , 3T3 Cells , Animals , Female , Gene Expression Regulation , Infertility, Male/enzymology , Male , Mice , Mice, Knockout , Oxidative Phosphorylation , Peptide Initiation Factors/chemistry , Protein Transport , Ribosomes/enzymology , Testis/enzymology , Testis/pathology
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