scholarly journals Enzyme-instructed morphological transition of the supramolecular assemblies of branched peptides

2020 ◽  
Vol 16 ◽  
pp. 2709-2718
Author(s):  
Dongsik Yang ◽  
Hongjian He ◽  
Bing Xu
Keyword(s):  
Enzymatic Reaction ◽  
Proteolytic Cleavage ◽  
Supramolecular Assemblies ◽  
Lysine Residue ◽  
Proteinase K ◽  
Morphological Transition ◽  
Cellular Environment ◽  
Branched Peptides ◽  
The Stability

Here, we report the use of an enzymatic reaction to cleave the branch off branched peptides for inducing the morphological transition of the assemblies of the peptides. The attachment of DEDDDLLI sequences to the ε-amine of the lysine residue of a tetrapeptide produces branched peptides that form micelles. Upon the proteolytic cleavage of the branch, catalyzed by proteinase K, the micelles turn into nanofibers. We also found that the acetylation of the N-terminal of the branch increased the stability of the branched peptides. Moreover, these branched peptides facilitate the delivery of the proteins into cells. This work contributes insights for the development of peptide supramolecular assemblies via enzymatic noncovalent synthesis in cellular environment.

scholarly journals Glycans in the intestinal peptide transporter PEPT1 contribute to function and protect from proteolysis

2017 ◽  
Vol 312 (6) ◽  
pp. G580-G591 ◽  
Author(s):  
Tamara Stelzl ◽  
Kerstin E. Geillinger-Kästle ◽  
Jürgen Stolz ◽  
Hannelore Daniel
Keyword(s):  
Protein Function ◽  
Proteolytic Cleavage ◽  
Glycosylation Site ◽  
Proteinase K ◽  
Peptide Transport ◽  
Peptide Transporter ◽  
Electrophysiological Recordings ◽  
Transport Velocity ◽  
Flux Measurements ◽  
The Individual

Despite the fact that many membrane proteins carry extracellular glycans, little is known about whether the glycan chains also affect protein function. We recently demonstrated that the proton-coupled oligopeptide transporter 1 (PEPT1) in the intestine is glycosylated at six asparagine residues (N50, N406, N439, N510, N515, and N532). Mutagenesis-induced disruption of the individual N-glycosylation site N50, which is highly conserved among mammals, was detected to significantly enhance the PEPT1-mediated inward transport of peptides. Here, we show that for the murine protein the inhibition of glycosylation at sequon N50 by substituting N50 with glutamine, lysine, or cysteine or by replacing S52 with alanine equally altered PEPT1 transport kinetics in oocytes. Furthermore, we provide evidence that the uptake of [14C]glycyl-sarcosine in immortalized murine small intestinal (MODE-K) or colonic epithelial (PTK-6) cells stably expressing the PEPT1 transporter N50Q is also significantly increased relative to the wild-type protein. By using electrophysiological recordings and tracer flux studies, we further demonstrate that the rise in transport velocity observed for PEPT1 N50Q is bidirectional. In line with these findings, we show that attachment of biotin derivatives, comparable in weight with two to four monosaccharides, to the PEPT1 N50C transporter slows down the transport velocity. In addition, our experiments provide strong evidence that glycosylation of PEPT1 confers resistance against proteolytic cleavage by proteinase K, whereas a remarkable intrinsic stability against trypsin, even in the absence of N-linked glycans, was detected. NEW & NOTEWORTHY This study highlights the role of N50-linked glycans in modulating the bidirectional transport activity of the murine peptide transporter PEPT1. Electrophysiological and tracer flux measurements in Xenopus oocytes have shown that removal of the N50 glycans increases the maximal peptide transport rate in the inward and outward directions. This effect could be largely reversed by replacement of N50 glycans with structurally dissimilar biotin derivatives. In addition, N-glycans were detected to stabilize PEPT1 against proteolytic cleavage.

Stabilization of the p53 transformation-related protein in mouse fibrosarcoma cell lines: effects of protein sequence and intracellular environment

1989 ◽  
Vol 9 (8) ◽  
pp. 3385-3392
Author(s):  
O Halevy ◽  
A Hall ◽  
M Oren
Keyword(s):  
Cell Lines ◽  
Primary Structure ◽  
P53 Protein ◽  
Cell Types ◽  
Stable Complex ◽  
Related Protein ◽  
Fibrosarcoma Cell ◽  
Mouse Tumors ◽  
Cellular Environment ◽  
The Stability

The transformation-related protein p53 is normally very labile. The stability of p53 is significantly increased in a number of fibrosarcoma cell lines derived from mouse tumors induced by treatment with physical or chemical agents. In many instances, p53 stabilization is correlated with the ability to form a stable complex with the heat shock protein cognate hsc70. We describe a line in which p53 is very stable yet has no detectable interaction with hsc70. The inability to form such a complex probably resides in the primary structure of the endogenous p53, since introduction of other p53 variants into those cells resulted in the appearance of a p53-hsc70 complex. The factors affecting p53 stability were investigated by stable transfection experiments. The results indicated that the primary structure of the p53 protein is a major determinant of its turnover rate; different p53 variants were degraded at distinct and characteristic rates in a number of transformed cell types. However, at least one p53 variant was degraded differently in nontransformed BALB/c-3T3 than in transformed fibrosarcoma cells, demonstrating that the specific cellular environment can also affect the stability of p53.

The stability of pseudopeptides bearing sulfoximines as chiral backbone modifying element towards proteinase K

2003 ◽  
Vol 13 (19) ◽  
pp. 3207-3211 ◽  
Author(s):  
Carsten Bolm ◽  
Dirk Müller ◽  
Christian Dalhoff ◽  
Christian P.R. Hackenberger ◽  
Elmar Weinhold
Keyword(s):  
Proteinase K ◽  
The Stability

Behaviour of RNA under hydrothermal conditions and the origins of life

2004 ◽  
Vol 3 (4) ◽  
pp. 301-309 ◽  
Author(s):  
Kunio Kawamura
Keyword(s):  
High Temperatures ◽  
Open Systems ◽  
Rna World ◽  
Enzymatic Reaction ◽  
Reaction Rates ◽  
Extensive Literature ◽  
Hydrothermal Conditions ◽  
Phosphodiester Bond ◽  
Rna Molecules ◽  
The Stability

The RNA world hypothesis and the hydrothermal origin of life hypothesis are contradictory to each other. Although it is considered that RNA molecules are too labile to maintain life-like systems at high temperatures and there is extensive literature on nucleic acid hydrolysis, the stability and the chemical evolution of RNA have not been sufficiently analysed from the viewpoint of hydrothermal reactions. Based on our experimental data concerning the stability and the prebiotic formation of RNA at high temperatures, two different aspects seem to be important for evaluating whether RNA molecules are too labile. First, the stability of RNA molecules should be evaluated from the comparison of the rate of formation and the rate of degradation of RNA in open systems. Our prebiotic reaction models of phosphodiester bond formation suggest that at high temperatures the accumulation of RNA may be possible. However, an RNA world entirely consisting of RNA molecules is unlikely to occur because the biologically important interactions are not effective for the bare RNA molecules at high temperatures. Second, since enzymes presently mediate most biological reactions, evaluation of the accumulation of RNA should be based on the comparison between the enzymatic and non-enzymatic reaction rates. Hence, the evaluation of the primitive enzymatic reaction rates at high temperatures has been attempted. There is a large difference between the present enzymatic reaction rates at 25–90 °C and the non-enzymatic reaction rates at high temperatures of 200–300 °C. It is thus possible that prebiotic enzyme-like assemblies could have facilitated the accumulation of RNA molecules at hydrothermal vent temperatures.

scholarly journals The fluorescent protein stability assay: an efficient method for monitoring intracellular protein stability

BioTechniques ◽  
2021 ◽  
Author(s):  
Armelle Roisin ◽  
Samuel Buchsbaum ◽  
Vincent Mocquet ◽  
Pierre Jalinot
Keyword(s):  
Protein Stability ◽  
Efficient Method ◽  
Fluorescent Protein ◽  
Specific Protein ◽  
Intracellular Protein ◽  
Fluorescent Reporter ◽  
Cellular Environment ◽  
Intracellular Proteins ◽  
Reporter Proteins ◽  
The Stability

The stability of intracellular proteins is highly variable, from a few minutes to several hours, and can be tightly regulated to respond to external and internal cellular environment changes. Several techniques can be used to study the stability of a specific protein, including pulse-chase labeling and blocking of translation. Another approach that has gained interest in recent years is fusing a protein of interest to a fluorescent reporter. In this report, the authors present a new version of this approach aimed at optimizing expression and comparison of the two reporter proteins. The authors show that the system works efficiently in various cells and can be useful for studying changes in protein stability and assessing the effects of drugs.

scholarly journals A fixative suitable for in situ hybridization histochemistry.

1993 ◽  
Vol 41 (6) ◽  
pp. 947-953 ◽  
Author(s):  
F Uehara ◽  
N Ohba ◽  
Y Nakashima ◽  
T Yanagita ◽  
M Ozawa ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Proteinase K ◽  
Optimal Time ◽  
Hybridization Histochemistry ◽  
Tissue Sections ◽  
In Situ Hybridization Histochemistry ◽  
Rna Probes ◽  
Labeled Rna ◽  
The Stability

We compared the morphology and stability of hybridization signals between paraffin sections of rat retina fixed with commonly used 4% paraformaldehyde/PBS and those fixed with a fixative containing glutaraldehyde in in situ hybridization histochemistry, using a digoxigenin-labeled RNA probe complementary for beta-galactoside alpha 2,6-sialyltransferase mRNA. Retinal detachment was frequently observed in the sections fixed with 4% paraformaldehyde-PBS, whereas the morphology was satisfactorily preserved in those fixed with either 0.5% glutaraldehyde, 4% paraformaldehyde-PBS, or 2.5% glutaraldehyde-PBS. Without glutaraldehyde, it was difficult to determine the most appropriate length of proteinase K digestion of tissue sections for facilitating probe penetration, since the optimal time for definite hybridization was variable among the retinal cells in heterogeneous layers. By addition of glutaraldehyde to paraformaldehyde or with glutaraldehyde alone, it was easy to establish the appropriate time for the unmasking procedure, since intense mRNA signals were constant throughout the retina by proteinase K digestion for more than 30-40 min. Using a fixative that causes stronger cross-linking (e.g., glutaraldehyde) is recommended to improve not only the morphology but also the stability of hybridization signals in in situ hybridization histochemistry with paraffin embedding and digoxigenin-labeled RNA probes.

scholarly journals Long-term stabilization of hydrogen peroxide by poly(vinyl alcohol) on paper-based analytical devices

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Tuchpongpuch Boonpoempoon ◽  
Wanida Wonsawat ◽  
Takashi Kaneta
Keyword(s):  
Hydrogen Peroxide ◽  
Vinyl Alcohol ◽  
Point Of Care ◽  
Enzymatic Reaction ◽  
Poly Vinyl Alcohol ◽  
Paper Substrate ◽  
Long Term Stability ◽  
Colorimetric Measurements ◽  
The Stability

Abstract Stabilizing reagents that can be deposited onto paper is an important issue for researchers who depend on paper-based analytical devices (PADs), because long-term stability of the devices is essential in point-of-care testing. Here, we found that poly(vinyl alcohol) (PVA) would stabilize hydrogen peroxide placed on a paper substrate following exposure to air. Horseradish peroxidase was employed as a sample in colorimetric measurements of PADs after hydrogen peroxide and 3,3′,5,5′-tetramethylbenzidine were deposited as substrates in an enzymatic reaction. The addition of PVA to hydrogen peroxide significantly suppressed its degradation. Concentrations of PVA that ranged from 0.5 to 2%, increased the duration of the stability of hydrogen peroxide, and the results for a PVA concentration of 1% approximated those of 2% PVA. Storage of the PADs at 4 °C in a refrigerator extended the stability of the hydrogen peroxide containing 2% PVA by as much as 30 days. The stability of hydrogen peroxide without PVA was degraded after one day under room temperature.

scholarly journals Biological functions of supramolecular assemblies of small molecules in the cellular environment

2021 ◽  
Author(s):  
Jingyu Wang ◽  
Hui Li ◽  
Bing Xu
Keyword(s):  
Small Molecules ◽  
Supramolecular Assemblies ◽  
Emergent Properties ◽  
Biological Functions ◽  
Therapeutic Approaches ◽  
Cellular Environment ◽  
Novel Therapeutic

Supramolecular assemblies of small molecules, exhibiting emergent properties, are becoming a new and dynamic molecular platform for biological functions and for developing novel therapeutic approaches.

scholarly journals Screening of additives to reduce grain damage risk on unhairing by proteinase K

2020 ◽  
Vol 2 (1) ◽  
Author(s):  
Mei Chen ◽  
Mingfang Jiang ◽  
He Li ◽  
Haiming Cheng
Keyword(s):  
Metal Ions ◽  
Collagen Fiber ◽  
Enzymatic Reaction ◽  
Hydrolytic Activity ◽  
Proteinase K ◽  
Organic Additives ◽  
Grain Damage ◽  
Potential Alternative ◽  
Bovine Hide ◽  
Damage Risk

Abstract Enzymatic unhairing is a cleaner strategy for leather-making. It is a potential alternative to the traditional hair-burning process. However, several shortcomings, such as uncontrolled enzymatic reaction, and risk of grain looseness and damage have restricted the broad application of enzymatic unhairing. In this work, metal ions and organic additives were screened for lessening the hydrolytic activity of proteinase K to collagen fiber. Then, the selected additives were applied to the enzymatic unhairing process for bovine hide. The results showed that a suitable concentration of metal ions (Cu (II), Fe (III) and Al (III)) and organic additives (salicylate, laurate, adipate, gallate and epicatechin (ECG)) could diminish approximately 35% of the hydrolytic activity of proteinase K to collagen fibers. Then, the additives were applied for the bovine hide enzymatic unhairing process. Hydroxyproline determination in the unhairing float shows that applying additives could reduce collagen hydrolysis. The morphology results showed that the grain damage could be significantly reduced with the addition of the screened additives in the proteinase K enzymatic unhairing system, whereas the addition of ECG and gallate significantly slowed down the unhairing speed. This outcome provides new potential to reduce the risk of grain damage in enzymatic unhairing process. Graphical abstract

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