Protein Synthesis -Translation and Regulation

There are twenty amino acids to choose from for protein synthesis, and a total of 64 triplet codons (four bases in triplet sequence = 4x4x4 combinations).

hormones, neurotransmitters - are packaged in secretory vesicles at the Golgi apparatus

As a matter of fact, protein biosynthesis is entirely controlled by RNA molecules including mRNA (genetic information), transfer RNA (tRNA) for translating the DNA code into amino acid code, and ribosomal RNA (rRNA) that provide the enzymatic linkage (chemical bond formation) of amino acids into proteins.


Human Physiology - Cell structure and function - EKU

An important open question of the study herein is whether inhibition of other glycosylation events occurring in the Golgi apparatus such as inhibition of transporters for UDP-galactose, UDP-N-acetylglucosamine, UDP-N-acetylgalactosamine, or Golgi apparatus glycosyltransferases lead to inhibition of nonglycoprotein synthesis, induction of ER stress, and inhibition of protein translation. A similar question pertains to inhibition of transport into the Golgi apparatus lumen by transporters for adenosine 3′-phosphate 5′-phosphosulfate as well as ATP. Although such studies will be easier to conduct in mammalian cell culture such as HeLa cells, extension of them to other eukaryotic cell culture systems and to multicellular organisms will be important.


Human Physiology - Cell structure and function

To determine whether or not under the above silencing conditions ER stress had occurred, we examined the phosphorylation (at Ser51) of eIF2α, a translation initiation factor that is activated in stressed cells and that, upon being phosphorylated, inhibits protein synthesis (). Total cell lysates were prepared from control and silenced HeLa cells, and phosphorylation of eIF2α was detected by Western blots. As can be seen in A (top and bottom panels), nucleotide sugar transporter silenced cells showed significant increased phosphorylation of eIF2α over controls. This was not the result of increased eIF2α protein levels as visualized with antibodies against the eIF2α protein (A, top panel). An additional control with antibodies against cdc2 showed that equal amounts of proteins had been added to each lane.

NRXN1 Gene - GeneCards | NRX1A Protein | NRX1A Antibody

The above results showed that silencing of transporters of CMP-sialic acid and GDP-fucose leads to reduced synthesis and secretion of both nonglycosylated and glycosylated proteins. We previously observed that conditions that induce ER stress up-regulate the transcription of the ER/Golgi nucleoside diphosphatase, uda-1, in C. elegans (). We hypothesized that as a result there is an increase in UMP that can be exchanged with UDP-glucose, relieving ER stress by favoring reglucosylation of unfolded proteins in the ER. Conversely we now hypothesized that inhibition of terminal glycosylation of proteins (sialylation and fucosylation) caused by silencing of either the Golgi apparatus CMP-sialic acid, GDP-fucose transporters, or both may induce ER stress.

All steps of protein synthesis easily ..

The broad consequences of nucleotide sugar transport inhibition on general protein glycosylation prompted us to study whether general protein synthesis had also been affected. Thus, HeLa cells in which the transporters of CMP-sialic acid and GDP-fucose had been silenced separately or simultaneously were radiolabeled with [35S]methionine and [35S]cysteine using the pulse-chase protocol previously described for . Aliquots of the cell lysate and growth medium were applied successively to WGA- and ConA-agarose columns. The flow-through of the latter column was then precipitated with tricholoroacetic acid, and the radioactivity of the washed pellet fraction, representing mostly nonglycoproteins, was determined, as was that of the glycoprotein fractions that bound to the columns. As can be seen in (left panels), there was a significant reduction in synthesis and secretion of nonglycoproteins in silenced cells, particularly in the cell lysate fraction. This unexpected result strongly suggests that the reduced synthesis and secretion previously observed with glycoproteins may actually be an even more general effect resulting from reduced synthesis and secretion of total cellular proteins. Reduction of synthesis and secretion of glycosylated proteins (fractions that bound to the above two columns) was also observed (, middle and right panels) in agreement with the above-described results shown with radiolabeled sugars (). However, we cannot rule out that the flow-through fraction in contains a small amount of glycosylated proteins that do not bind to the above lectin columns.