scholarly journals Multiple prebiotic metals mediate translation

2018 ◽  
Author(s):  
Marcus S. Bray ◽  
Timothy K. Lenz ◽  
Jay William Haynes ◽  
Jessica C. Bowman ◽  
Anton S. Petrov ◽  
...  
Keyword(s):  
Messenger Rna ◽  
Tertiary Structure ◽  
Cell Function ◽  
Living Organism ◽  
Translation System ◽  
Ancestral State ◽  
Functional Protein ◽  
Functional Roles ◽  
And Function

ABSTRACTToday, Mg2+is an essential cofactor with diverse structural and functional roles in life’s oldest macromolecular machine, the translation system. We tested whether ancient Earth conditions (low O2, high Fe2+, high Mn2+) can revert the ribosome to a functional ancestral state. First, SHAPE (Selective 2’HydroxylAcylation analyzed byPrimerExtension) was used to compare the effect of Mg2+, Fe2+, and Mn2+on the tertiary structure of rRNA. Then, we usedin vitrotranslation reactions to test whether Fe2+or Mn2+could mediate protein production, and quantified ribosomal metal content. We found that: (i) Mg2+, Fe2+, and Mn2+had strikingly similar effects on rRNA folding; (ii) Fe2+and Mn2+can replace Mg2+as the dominant divalent cation during translation of mRNA to functional protein; (iii) Fe and Mn associate extensively with the ribosome. Given that the translation system originated and matured when Fe2+and Mn2+were abundant, these findings suggest that Fe2+and Mn2+played a role in early ribosomal evolution.SIGNIFICANCERibosomes are found in every living organism where they are responsible for the translation of messenger RNA into protein. The ribosome’s centrality to cell function is underscored by its evolutionary conservation; the core structure has changed little since its inception ~4 billion years ago when ecosystems were anoxic and metal-rich. The ribosome is a model system for the study of bioinorganic chemistry, owing to the many highly coordinated divalent metal cations that are essential to its function. We studied the structure, function, and cation content of the ribosome under early Earth conditions (low O2, high Fe2+, high Mn2+). Our results expand the roles of Fe2+and Mn2+in ancient and extant biochemistry as cofactors for ribosomal structure and function.

scholarly journals Multiple prebiotic metals mediate translation

2018 ◽  
Vol 115 (48) ◽  
pp. 12164-12169 ◽  
Author(s):  
Marcus S. Bray ◽  
Timothy K. Lenz ◽  
Jay William Haynes ◽  
Jessica C. Bowman ◽  
Anton S. Petrov ◽  
...  
Keyword(s):  
Metal Content ◽  
Protein Production ◽  
Tertiary Structure ◽  
In Vitro Translation ◽  
Translation System ◽  
Ancestral State ◽  
Functional Protein ◽  
Functional Roles ◽  
Shape Selective

Today, Mg2+is an essential cofactor with diverse structural and functional roles in life’s oldest macromolecular machine, the translation system. We tested whether ancient Earth conditions (low O2, high Fe2+, and high Mn2+) can revert the ribosome to a functional ancestral state. First, SHAPE (selective 2′-hydroxyl acylation analyzed by primer extension) was used to compare the effect of Mg2+, Fe2+, and Mn2+on the tertiary structure of rRNA. Then, we used in vitro translation reactions to test whether Fe2+or Mn2+could mediate protein production, and quantified ribosomal metal content. We found that (i) Mg2+, Fe2+, and Mn2+had strikingly similar effects on rRNA folding; (ii) Fe2+and Mn2+can replace Mg2+as the dominant divalent cation during translation of mRNA to functional protein; and (iii) Fe and Mn associate extensively with the ribosome. Given that the translation system originated and matured when Fe2+and Mn2+were abundant, these findings suggest that Fe2+and Mn2+played a role in early ribosomal evolution.

scholarly journals Subpopulations of soluble, misfolded proteins commonly bypass chaperones: How it happens at the molecular level

2021 ◽  
Author(s):  
Ritaban Halder ◽  
Daniel A. Nissley ◽  
Ian Sitarik ◽  
Edward P. O’Brien
Keyword(s):  
Tertiary Structure ◽  
Molecular Level ◽  
Meta Analysis ◽  
Hydrophobic Surface ◽  
Misfolded Proteins ◽  
Similar Degree ◽  
Functional Protein ◽  
Folded Proteins ◽  
And Function

ABSTRACTSubpopulations of soluble, misfolded proteins can bypass chaperones within cells. The scope of this phenomenon and the lifetimes of these states have not been experimentally quantified, and how such misfolding happens at the molecular level is poorly understood. We address the first issue through a meta-analysis of the experimental literature. We find that in all quantitative protein refolding-function studies, there is always a subpopulation of soluble but misfolded and less-functional protein that does not fold in the presence of one or more chaperones. This subpopulation ranges from 8% to 50% of the soluble protein molecules in solution. Fitting the experimental time traces to a kinetic model, we find these chaperone-bypassing misfolded states take months or longer to fold and function in the presence of different chaperones. We next addressed how, at the molecular level, some misfolded proteins can evade chaperones by simulating six different proteins interacting with E. coli’s GroEL and HtpG chaperones when those proteins are in folded, unfolded, or long-lived, soluble, misfolded states. We observe that both chaperones strongly bind the unfolded state and weakly bind the folded and misfolded states to a similar degree. Thus, these chaperones cannot distinguish between the folded and long-lived misfolded states of these proteins. A structural analysis reveals the misfolded states are highly similar to the native state – having a similar size, amount of exposed hydrophobic surface area, and level of tertiary structure formation. These results demonstrate that in vitro it is common for appreciable subpopulations of proteins to remain misfolded, soluble, and evade the refolding action of chaperones for very long times. Further, these results suggest that this happens because these misfolded subpopulations are near-native and therefore interact with chaperones to a similar extent as properly folded proteins. More broadly, these results indicate a mechanism in which long-time scale changes in protein structure and function can persist in cells because some protein’s non-native states can bypass components of the proteostasis machinery.TEASERNear-native, misfolded protein conformations explain why some soluble proteins fail to refold in the presence of chaperones.

scholarly journals The STAGA Subunit ADA2b Is an Important Regulator of Human GCN5 Catalysis

2008 ◽  
Vol 29 (1) ◽  
pp. 266-280 ◽  
Author(s):  
Armin M. Gamper ◽  
Jaehoon Kim ◽  
Robert G. Roeder
Keyword(s):  
Saga Complex ◽  
Histone Tails ◽  
Functional Roles ◽  
Enzymatic Function ◽  
Core Histones ◽  
Human Proteins ◽  
Important Regulator ◽  
And Function

ABSTRACT Human STAGA is a multisubunit transcriptional coactivator containing the histone acetyltransferase GCN5L. Previous studies of the related yeast SAGA complex have shown that the yeast Gcn5, Ada2, and Ada3 components form a heterotrimer that is important for the enzymatic function of SAGA. Here, we report that ADA2a and ADA2b, two human homologues of yeast Ada2, each have the ability to form a heterotrimer with ADA3 and GCN5L but that only the ADA2b homologue is found in STAGA. By comparing the patterns of acetylation of several substrates, we found context-dependent requirements for ADA2b and ADA3 for the efficient acetylation of histone tails by GCN5. With human proteins, unlike yeast proteins, the acetylation of free core histones by GCN5 is unaffected by ADA2b or ADA3. In contrast, the acetylation of mononucleosomal substrates by GCN5 is enhanced by ADA2b, with no significant additional effect of ADA3, and the efficient acetylation of nucleosomal arrays (chromatin) by GCN5 requires both ADA2b and ADA3. Thus, ADA2b and ADA3 appear to act at two different levels of histone organization within chromatin to facilitate GCN5 function. Interestingly, although ADA2a forms a complex(es) with GCN5 and ADA3 both in vitro and in vivo, ADA2a-containing complexes are unable to acetylate nucleosomal H3. We have also shown the preferential recruitment of ADA2b, relative to ADA2a, to p53-dependent genes. This finding indicates that the previously demonstrated presence and function of GCN5 on these promoters reflect the action of STAGA and that the ADA2a and ADA2b paralogues have nonredundant functional roles.

scholarly journals Low c-kit expression of cultured mast cells of mi/mi genotype may be involved in their defective responses to fibroblasts that express the ligand for c-kit

Blood ◽  
1992 ◽  
Vol 80 (6) ◽  
pp. 1454-1462 ◽  
Author(s):  
Y Ebi ◽  
Y Kanakura ◽  
T Jippo-Kanemoto ◽  
T Tsujimura ◽  
T Furitsu ◽  
...  
Keyword(s):  
Mast Cell ◽  
Mast Cells ◽  
Tyrosine Phosphorylation ◽  
Messenger Rna ◽  
Tissue Type ◽  
Phenotypic Change ◽  
3T3 Fibroblasts ◽  
Mouse Mast Cell ◽  
And Function

Abstract Mutant mice of mi/mi genotype are osteopetrotic and deficient in tissue mast cells due to a defect in osteoclasts and mast cells. In an effort to further understand the mechanisms behind why mi/mi mouse-derived cultured mast cells (mi/mi-CMC) responded to interleukin-3 (IL-3), but not to the proliferative stimuli presented by fibroblasts, mi/mi-CMC and congenic normal (+/+) mouse-derived CMC (+/+-CMC), both of which expressed the phenotypic characteristics of immature mast cells, were cocultured with Swiss albino/3T3 fibroblasts in a medium containing IL- 3. In the in vitro CMC/fibroblast coculture, mi/mi-CMC did not acquire the phenotypes of connective tissue-type mast cells (CTMC), while +/+- CMC did. In addition, attachment of mi/mi-CMC to the fibroblasts was found to be significantly lower than that of +/+-CMC. Because the interaction of c-kit product with its ligand (stem cell factor [SCF]) is known to play an important role not only in proliferation and differentiation of mast cells but also in attachment of CMC to fibroblasts, the expression and function of c-kit were investigated in mi/mi-CMC and +/+-CMC. Recombinant rat SCF (rrSCF164) induced a dose- dependent proliferation of +/+-CMC. Also, rrSCF164 induced +/+-CMC to acquire the phenotypes of CTMC in the medium containing IL-3. By contrast, rrSCF164 did not stimulate the proliferation of mi/mi-CMC nor induce a phenotypic change of the cells from immature mast cells to mature, CTMC-like mast cells. Immunoblotting with antiphosphotyrosine antibody showed that rrSCF164 induced considerable tyrosine phosphorylation of 145- to 165-Kd protein, the product of c-kit, in +/+- CMC, whereas tyrosine phosphorylation of the protein was barely detectable in mi/mi-CMC. Northern blot and flow cytometry analyses showed that mi/mi-CMC expressed much less c-kit at both protein and message levels than +/+-CMC. Further, mi/mi-CMC were found to differ from +/+-CMC in the expression of mouse mast cell protease-6 (MMCP-6) and MMCP-2 messenger RNA transcripts. These results suggest that the gene product of the mi locus may be important in regulating the expression of gene products such as c-kit, and that mast cell deficiency of mi/mi mice appears to be due, at least in part, to impaired signaling through the c-kit receptor because of the low c-kit expression.

scholarly journals Circulating miRNA 887 is differentially expressed in ARDS and modulates endothelial function

2020 ◽  
Vol 318 (6) ◽  
pp. L1261-L1269 ◽  
Author(s):  
Andrew J. Goodwin ◽  
Pengfei Li ◽  
Perry V. Halushka ◽  
James A. Cook ◽  
Aman S. Sumal ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Function ◽  
In Silico Analysis ◽  
Leukocyte Migration ◽  
Differentially Expressed ◽  
Expression Of Genes ◽  
Cell Gene Expression ◽  
And Function ◽  
The Impact

Circulating microRNAs (miRNAs) can be taken up by recipient cells and have been recently associated with the acute respiratory distress syndrome (ARDS). Their role in host predisposition to the syndrome is unknown. The objective of the study was to identify circulating miRNAs associated with the development of sepsis-related ARDS and examine their impact on endothelial cell gene expression and function. We determined miRNA levels in plasma collected from subjects during the first 24 h of admission to a tertiary intensive care unit for sepsis. A miRNA that was differentially expressed between subjects who did and did not develop ARDS was identified and was transfected into human pulmonary microvascular endothelial cells (HPMECs). RNA sequencing, in silico analysis, cytokine expression, and leukocyte migration assays were used to determine the impact of this miRNA on gene expression and cell function. In two cohorts, circulating miR-887-3p levels were elevated in septic patients who developed ARDS compared with those who did not. Transfection of miR-887-3p into HPMECs altered gene expression, including the upregulation of several genes previously associated with ARDS (e.g., CXCL10, CCL5, CX3CL1, VCAM1, CASP1, IL1B, IFNB, and TLR2), and activation of cellular pathways relevant to the response to infection. Functionally, miR-887-3p increased the endothelial release of chemokines and facilitated trans-endothelial leukocyte migration. Circulating miR-887-3p is associated with ARDS in critically ill patients with sepsis. In vitro, miR-887-3p regulates the expression of genes relevant to ARDS and neutrophil tracking. This miRNA may contribute to ARDS pathogenesis and could represent a novel therapeutic target.

scholarly journals PRMT5 Is Upregulated in Activated T Cells and Is a Novel Therapeutic Target for Acute Gvhd

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 2034-2034
Author(s):  
Parvathi Ranganathan ◽  
Katiri Snyder ◽  
Nina Zizter ◽  
Hannah K. Choe ◽  
Robert A Baiocchi ◽  
...  
Keyword(s):  
Cell Cycle ◽  
T Cells ◽  
T Cell ◽  
Western Blot ◽  
Cell Function ◽  
T Cell Proliferation ◽  
Activated T Cells ◽  
And Function

Abstract Introduction: Acute graft-versus-host disease (aGVHD), a T cell-mediated immunological disorder is the leading cause of non-relapse mortality in patients receiving allogeneic bone marrow transplants. Protein arginine methyltransferase 5 (PRMT5) catalyzes symmetric dimethylation (me2s) of arginine (R) residues on histones (primarily H3R8 and H3R4) and other proteins. PRMT5 is overexpressed in many leukemias and lymphomas, and epigenetic changes driven by PRMT5 lead to repression of tumor suppressors and promote growth and survival of cancer cells. Recently it was shown that T cells are sensitive to R-methylation and PRMT5 promotes activation of memory T helper cells. Here we investigate: 1) mechanisms by which PRMT5 regulates T cell function; and 2) PRMT5 inhibition as a therapeutic strategy for aGVHD. Materials and Methods: Splenic T cells were isolated from lethally irradiated B6D2F1 mice that received either T cell depleted bone marrow (TCD-BM) or TCD-BM with C57/BL6 (B6) allogeneic splenocytes on day 21 post-transplant. In vitro activation of B6 T cells was achieved with CD3/CD28 Dynabeads or co-culture with allogeneic BM-derived dendritic cells. PRMT5 expression (RT-PCR, western blot) and function (H3R8me2s western blot) were evaluated. PRT220, a novel inhibitor of PRMT5, was used to evaluate PRMT5 inhibition on T cell function in vitro and in vivo. We assessed T cell proliferation (Cell Trace Violet, Ki67), apoptosis (Annexin V), cytokine secretion (ELISA, flow cytometry), cell cycle (PI incorporation), and cell signaling (western blot). Lethally irradiated F1 recipients received TCD-BM only (10x106 cells) or TCD-BM + B6 splenocytes (20 x 106). Recipients of allogeneic splenocytes were treated with PRT220 (2mg/kg) or vehicle by oral gavage once weekly starting day 7 post-transplant. Mice were monitored for survival and clinical aGVHD scores. Results: PRMT5 expression and function is upregulated following T cell activation. Inhibition of PRMT5 reduces T cell proliferation and IFN-g secretion. PRMT5 inhibition in CD3/CD28 stimulated T cells results in disruption of multiple histone epigenetic marks, cell-cycle progression (via G1 arrest) and perturbation of ERK-MAPK signaling cascades. Finally, administration of PRT220 resulted in significantly prolonging the survival of allo-transplanted recipient mice (median survival, PRT220 vs. vehicle, 36.5 vs. 26 days, p=0.01). PRT220-treated recipients also exhibited significant lower aGVHD clinical (p<0.05), pathological scores (p<0.05) and lower serum TNF-a (p<0.05) and IFN-g (p<0.05) than vehicle-treated recipients. Conclusions: PRMT5 expression and function are upregulated in activated T cells. Inhibition of PRMT5 function using a novel and specific small-molecule inhibitor, PRT220, down-regulates T cells proliferative and effector response, induces cell-cycle arrest and perturbs signaling pathways. PRT220 shows potent biological activity in vivo by reducing aGVHD clinical severity and significantly prolonging survival in mouse models of aGVHD. Therefore, PRMT5 is a novel and druggable target for aGVHD. Disclosures No relevant conflicts of interest to declare.

scholarly journals Specific effects of antitumor active norspermidine on the structure and function of DNA

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Takashi Nishio ◽  
Yuko Yoshikawa ◽  
Chwen-Yang Shew ◽  
Naoki Umezawa ◽  
Tsunehiko Higuchi ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Function ◽  
Specific Effect ◽  
Underlying Mechanism ◽  
Chemical Structures ◽  
Specific Effects ◽  
Methylene Groups ◽  
And Function ◽  
Dna Measurements

Abstract We compared the effects of trivalent polyamines, spermidine (SPD) and norspermidine (NSPD), a chemical homologue of SPD, on the structure of DNA and gene expression. The chemical structures of SPD and NSPD are different only with the number of methylene groups between amine groups, [N-3-N-4-N] and [N-3-N-3-N], respectively. SPD plays vital roles in cell function and survival, including in mammals. On the other hand, NSPD has antitumor activity and is found in some species of plants, bacteria and algae, but not in humans. We found that both polyamines exhibit biphasic effect; enhancement and inhibition on in vitro gene expression, where SPD shows definitely higher potency in enhancement but NSPD causes stronger inhibition. Based on the results of AFM (atomic force microscopy) observations together with single DNA measurements with fluorescence microscopy, it becomes clear that SPD tends to align DNA orientation, whereas NSPD induces shrinkage with a greater potency. The measurement of binding equilibrium by NMR indicates that NSPD shows 4–5 times higher affinity to DNA than SPD. Our theoretical study with Monte Carlo simulation provides the insights into the underlying mechanism of the specific effect of NSPD on DNA.

scholarly journals Exhausted CD8+ T cells exhibit low and strongly inhibited TCR signaling during chronic LCMV infection

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Ioana Sandu ◽  
Dario Cerletti ◽  
Manfred Claassen ◽  
Annette Oxenius
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Function ◽  
Viral Infections ◽  
Cell Receptor ◽  
Target Cells ◽  
Tcr Signaling ◽  
And Function

Abstract Chronic viral infections are often associated with impaired CD8+ T cell function, referred to as exhaustion. Although the molecular and cellular circuits involved in CD8+ T cell exhaustion are well defined, with sustained presence of antigen being one important parameter, how much T cell receptor (TCR) signaling is actually ongoing in vivo during established chronic infection is unclear. Here, we characterize the in vivo TCR signaling of virus-specific exhausted CD8+ T cells in a mouse model, leveraging TCR signaling reporter mice in combination with transcriptomics. In vivo signaling in exhausted cells is low, in contrast to their in vitro signaling potential, and despite antigen being abundantly present. Both checkpoint blockade and adoptive transfer of naïve target cells increase TCR signaling, demonstrating that engagement of co-inhibitory receptors curtails CD8+ T cell signaling and function in vivo.

Effect of Epidermal Growth Factor on Adult Rat Hepatocyte Monolayer Cultures

1980 ◽  
Vol 38 ◽  
pp. 778-779
Author(s):  
W. A. Samsonoff ◽  
R. Jansing
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Cell Function ◽  
Parenchymal Cell ◽  
Male Rats ◽  
Monolayer Cultures ◽  
Epidermal Growth ◽  
And Function ◽  
Hepatocyte Monolayer

Primary hepatocyte monolayer cultures have proven useful for in vitro study of parenchymal cell function. Unfortunately these cultures lose liver function rapidly and are often short-lived. Modified subtrates (1,2) can overcome some of these problems, but they produce fewer viable cells. Specific media supplements can also prolong cellular function. Epidermal growth factor (EGF) for example, significantly affects the structure and function of cells in culture (3). In this study we determined the effect of EGF on the ultrastructure and function of hepatocytes from Fisher 344 adult male rats. They were maintained as primary monolayer cultures in Leibovitz L-15 medium with supplements.

Sign in / Sign up

Export Citation Format

Share Document