scholarly journals Defining the current scope and limitations of dual noncanonical amino acid mutagenesis in mammalian cells

2017 ◽  
Vol 8 (10) ◽  
pp. 7211-7217 ◽  
Author(s):  
Yunan Zheng ◽  
Partha Sarathi Addy ◽  
Raja Mukherjee ◽  
Abhishek Chatterjee
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Mammalian Cells ◽  
Noncanonical Amino Acids ◽  
Amino Acid Mutagenesis ◽  
Noncanonical Amino Acid

We systematically evaluate potential platforms for site-specifically incorporating two distinct noncanonical amino acids into proteins expressed in mammalian cells with optimal fidelity and efficiency – a technology that will have many enabling applications.

scholarly journals Expanding the Scope of Single- and Double-Noncanonical Amino Acid Mutagenesis in Mammalian Cells Using Orthogonal Polyspecific Leucyl-tRNA Synthetases

Biochemistry ◽  
2017 ◽  
Vol 57 (4) ◽  
pp. 441-445 ◽  
Author(s):  
Yunan Zheng ◽  
Raja Mukherjee ◽  
Melissa A. Chin ◽  
Peter Igo ◽  
Martin J. Gilgenast ◽  
...  
Keyword(s):  
Amino Acid ◽  
Mammalian Cells ◽  
Trna Synthetases ◽  
Amino Acid Mutagenesis ◽  
Noncanonical Amino Acid

scholarly journals A Genetically Encoded Picolyl Azide for Improved Live Cell Copper Click Labeling

2021 ◽  
Vol 9 ◽  
Author(s):  
Birthe Meineke ◽  
Johannes Heimgärtner ◽  
Alexander J. Craig ◽  
Michael Landreh ◽  
Lindon W. K. Moodie ◽  
...  
Keyword(s):  
Amino Acids ◽  
Mammalian Cells ◽  
Stop Codon ◽  
Bioorthogonal Chemistry ◽  
Bioorthogonal Reaction ◽  
Noncanonical Amino Acids ◽  
Selective Reactivity ◽  
Direct Incorporation ◽  
Chelating Groups ◽  
Amber Suppression

Bioorthogonal chemistry allows rapid and highly selective reactivity in biological environments. The copper-catalyzed azide–alkyne cycloaddition (CuAAC) is a classic bioorthogonal reaction routinely used to modify azides or alkynes that have been introduced into biomolecules. Amber suppression is an efficient method for incorporating such chemical handles into proteins on the ribosome, in which noncanonical amino acids (ncAAs) are site specifically introduced into the polypeptide in response to an amber (UAG) stop codon. A variety of ncAA structures containing azides or alkynes have been proven useful for performing CuAAC chemistry on proteins. To improve CuAAC efficiency, biologically incorporated alkyne groups can be reacted with azide substrates that contain copper-chelating groups. However, the direct incorporation of copper-chelating azides into proteins has not been explored. To remedy this, we prepared the ncAA paz-lysine (PazK), which contains a picolyl azide motif. We show that PazK is efficiently incorporated into proteins by amber suppression in mammalian cells. Furthermore, PazK-labeled proteins show improved reactivity with alkyne reagents in CuAAC.

Na(+)-independent transport (uniport) of amino acids and glucose in mammalian cells.

1994 ◽  
Vol 196 (1) ◽  
pp. 93-108
Author(s):  
D K Kakuda ◽  
C L MacLeod
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Mammalian Cells ◽  
Glucose Transporter ◽  
Sequence Similarity ◽  
Amino Acid Sequence Similarity ◽  
Amino Acid Transporters ◽  
Cationic Amino Acid ◽  
Transporter Family ◽  
Share Amino Acid Sequence

Recent advances have made possible the isolation of the genes and their cDNAs encoding Na(+)-independent amino acid transporters. Two classes of amino acid 'uniporters' have been isolated. One class contains the mCAT (murine cationic amino acid transporter) gene family that encodes proteins predicted to span the membrane 12-14 times and exhibits structural properties similar to the GLUT (glucose transporter) family and to other well-known transporters. The other class consists of two known genes, rBAT (related to B system amino acid transporters) and 4F2hc, that share amino acid sequence similarity with alpha-amylases and alpha-glucosidases. They are type II glycoproteins predicted to span the membrane only once, yet they mediate the Na(+)-independent transport of cationic and zwitterionic amino acids in Xenopus oocytes. Mutations in the human rBAT gene have been identified by Palacín and his co-workers in several families suffering from a heritable form of cystinuria. This important finding clearly establishes a key role for rBAT in cystine transport. The two classes of amino acid transporters are compared with the well-studied GLUT family of Na(+)-independent glucose transporters.

Cloning and functional characterization of a new subtype of the amino acid transport system N

2001 ◽  
Vol 281 (6) ◽  
pp. C1757-C1768 ◽  
Author(s):  
Takeo Nakanishi ◽  
Ramesh Kekuda ◽  
You-Jun Fei ◽  
Takahiro Hatanaka ◽  
Mitsuru Sugawara ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Transport System ◽  
Amino Acid Transport ◽  
Mammalian Cells ◽  
Functional Characterization ◽  
Isobutyric Acid ◽  
Xenopus Laevis Oocytes ◽  
Acid Transport ◽  
Amino Acid Transport System

We have cloned a new subtype of the amino acid transport system N2 (SN2 or second subtype of system N) from rat brain. Rat SN2 consists of 471 amino acids and belongs to the recently identified glutamine transporter gene family that consists of system N and system A. Rat SN2 exhibits 63% identity with rat SN1. It also shows considerable sequence identity (50–56%) with the members of the amino acid transporter A subfamily. In the rat, SN2 mRNA is most abundant in the liver but is detectable in the brain, lung, stomach, kidney, testis, and spleen. When expressed in Xenopus laevis oocytes and in mammalian cells, rat SN2 mediates Na+-dependent transport of several neutral amino acids, including glycine, asparagine, alanine, serine, glutamine, and histidine. The transport process is electrogenic, Li+tolerant, and pH sensitive. The transport mechanism involves the influx of Na+ and amino acids coupled to the efflux of H+, resulting in intracellular alkalization. Proline, α-(methylamino)isobutyric acid, and anionic and cationic amino acids are not recognized by rat SN2.

Targeting motifs and functional parameters governing the assembly of connexins into gap junctions

2000 ◽  
Vol 349 (1) ◽  
pp. 281-287 ◽  
Author(s):  
Patricia E. M. MARTIN ◽  
James STEGGLES ◽  
Claire WILSON ◽  
Shoeb AHMAD ◽  
W. Howard EVANS
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Gap Junctions ◽  
Gap Junction ◽  
Mammalian Cells ◽  
Fluorescent Protein ◽  
Lucifer Yellow ◽  
Amino Acid Sequences ◽  
Amino Acid Residues ◽  
Green Fluorescent

To study the assembly of gap junctions, connexin-green-fluorescent-protein (Cx-GFP) chimeras were expressed in COS-7 and HeLa cells. Cx26- and Cx32-GFP were targeted to gap junctions where they formed functional channels that transferred Lucifer Yellow. A series of Cx32-GFP chimeras, truncated from the C-terminal cytoplasmic tail, were studied to identify amino acid sequences governing targeting from intracellular assembly sites to the gap junction. Extensive truncation of Cx32 resulted in failure to integrate into membranes. Truncation of Cx32 to residue 207, corresponding to removal of most of the 78 amino acids on the cytoplasmic C-terminal tail, led to arrest in the endoplasmic reticulum and incomplete oligomerization. However, truncation to amino acid 219 did not impair Cx oligomerization and connexon hemichannels were targeted to the plasma membrane. It was concluded that a crucial gap-junction targeting sequence resides between amino acid residues 207 and 219 on the cytoplasmic C-terminal tail of Cx32. Studies of a Cx32E208K mutation identified this as one of the key amino acids dictating targeting to the gap junction, although oligomerization of this site-specific mutation into hexameric hemichannels was relatively unimpaired. The studies show that expression of these Cx-GFP constructs in mammalian cells allowed an analysis of amino acid residues involved in gap-junction assembly.

scholarly journals The heterodimeric amino acid transporter 4F2hc/y+LAT2 mediates arginine efflux in exchange with glutamine

2000 ◽  
Vol 349 (3) ◽  
pp. 787-795 ◽  
Author(s):  
Angelika BRÖER ◽  
Carsten A. WAGNER ◽  
Florian LANG ◽  
Stefan BRÖER
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Mammalian Cells ◽  
Substrate Inhibition ◽  
Cell Types ◽  
Xenopus Laevis Oocytes ◽  
Cationic Amino Acid ◽  
New Family ◽  
Arginine Uptake ◽  
Intracellular Binding

The cationic amino acid arginine, due to its positive charge, is usually accumulated in the cytosol. Nevertheless, arginine has to be released by a number of cell types, e.g. kidney cells, which supply other organs with this amino acid, or the endothelial cells of the blood–brain barrier which release arginine into the brain. Arginine release in mammalian cells can be mediated by two different transporters, y+LAT1 and y+LAT2. For insertion into the plasma membrane, these transporters have to be associated with the type-II membrane glycoprotein 4F2hc [Torrents, Estevez, Pineda, Fernandez, Lloberas, Shi, Zorzano and Palacin (1998) J. Biol. Chem. 273, 32437–32445]. The present study elucidates the function and distribution of y+LAT2. In contrast to y+LAT1, which is expressed mainly in kidney epithelial cells, lung and leucocytes, y+LAT2 has a wider tissue distribution, including brain, heart, testis, kidney, small intestine and parotis. When co-expressed with 4F2hc in Xenopus laevis oocytes, y+LAT2 mediated uptake of arginine, leucine and glutamine. Arginine uptake was inhibited strongly by lysine, glutamate, leucine, glutamine, methionine and histidine. Mutual inhibition was observed when leucine or glutamine was used as substrate. Inhibition of arginine uptake by neutral amino acids depended on the presence of Na+, which is a hallmark of y+LAT-type transporters. Although arginine transport was inhibited strongly by glutamate, this anionic amino acid was only weakly transported by 4F2hc/y+LAT2. Amino acid transport via 4F2hc/y+LAT2 followed an antiport mechanism similar to the other members of this new family. Only preloaded arginine could be released in exchange for extracellular amino acids, whereas marginal release of glutamine or leucine was observed under identical conditions. These results indicated that arginine has the highest affinity for the intracellular binding site and that arginine release may be the main physiological function of this transporter.

The role of amino acid transporters in inherited and acquired diseases

2011 ◽  
Vol 436 (2) ◽  
pp. 193-211 ◽  
Author(s):  
Stefan Bröer ◽  
Manuel Palacín
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Mammalian Cells ◽  
Neuronal Excitability ◽  
Tumour Progression ◽  
Building Blocks ◽  
Amino Acid Uptake ◽  
Whole Body ◽  
Amino Acid Transporters ◽  
Acid Uptake

Amino acids are essential building blocks of all mammalian cells. In addition to their role in protein synthesis, amino acids play an important role as energy fuels, precursors for a variety of metabolites and as signalling molecules. Disorders associated with the malfunction of amino acid transporters reflect the variety of roles that they fulfil in human physiology. Mutations of brain amino acid transporters affect neuronal excitability. Mutations of renal and intestinal amino acid transporters affect whole-body homoeostasis, resulting in malabsorption and renal problems. Amino acid transporters that are integral parts of metabolic pathways reduce the function of these pathways. Finally, amino acid uptake is essential for cell growth, thereby explaining their role in tumour progression. The present review summarizes the involvement of amino acid transporters in these roles as illustrated by diseases resulting from transporter malfunction.

scholarly journals High-Yield, Zero-Leakage Expression System with a Translational Switch Using Site-Specific Unnatural Amino Acid Incorporation

2013 ◽  
Vol 80 (5) ◽  
pp. 1718-1725 ◽  
Author(s):  
Masaomi Minaba ◽  
Yusuke Kato
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Unnatural Amino Acids ◽  
Mammalian Cells ◽  
Expression System ◽  
Trna Synthetase ◽  
Unnatural Amino Acid ◽  
Translational Efficiency ◽  
Site Specific ◽  
Content Type

ABSTRACTSynthetic biologists construct complex biological circuits by combinations of various genetic parts. Many genetic parts that are orthogonal to one another and are independent of existing cellular processes would be ideal for use in synthetic biology. However, our toolbox is still limited with respect to the bacteriumEscherichia coli, which is important for both research and industrial use. The site-specific incorporation of unnatural amino acids is a technique that incorporates unnatural amino acids into proteins using a modified exogenous aminoacyl-tRNA synthetase/tRNA pair that is orthogonal to any native pairs in a host and is independent from other cellular functions. Focusing on the orthogonality and independency that are suitable for the genetic parts, we designed novel AND gate and translational switches using the unnatural amino acid 3-iodo-l-tyrosine incorporation system inE. coli. A translational switch was turned on after addition of 3-iodo-l-tyrosine in the culture medium within minutes and allowed tuning of switchability and translational efficiency. As an application, we also constructed a gene expression system that produced large amounts of proteins under induction conditions and exhibited zero-leakage expression under repression conditions. Similar translational switches are expected to be applicable also for eukaryotes such as yeasts, nematodes, insects, mammalian cells, and plants.

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