The Cell and Protein Synthesis/Secretion
Heparan sulfate proteoglycans: structure, protein interactions and cell signaling
Cell type-specific control of protein synthesis and proliferation ..
E177 has been identified as an invariant amino acid across the RIP family. It lies within the active site of RTA and is known to be a key catalytic residue (). In the present study, conversion of E177 to lysine (E177K) produced mutants that exhibited virtually no detectable depurination activity and failed to inhibit protein synthesis, induce apoptosis or activate signaling of the JNK and p38 cascades. This agrees with results obtained with other systems, e.g., this mutation led to total inactivation of the enzyme in an in vitro translation assay () and allowed growth in yeast (, ). Pre- and mature E177K were expressed at the highest level relative to all other constructs. Interestingly, in a study where amino acids were systematically deleted to determine their role in RTA activity, an inverse correlation was observed between expression in E. coli and enzymatic activity in vitro (). This suggests that the very high level of E177K expression may be related to complete lack of biological activity of the mutant protein. These results are also consistent with previous studies in yeast () and suggest that greater enzymatic activity of RTA will lead to higher translation inhibition and reduced protein accumulation.
Post‐translational processing and trafficking of secreted and transmembrane proteins in neurons: (a) During and after translation, secreted and transmembrane proteins receive post‐translational modifications (text, left) as they pass through the endoplasmic reticulum (ER), Golgi apparatus and ‐Golgi network (TGN). Finally, vesicles containing the proteins bud from the TGN and are transported to their final destination. Targeting mechanisms guide the vesicles to the axon or dendrites and then to the correct area within each where they are secreted or inserted into the plasma membrane. In a process called transcytosis (centre), some axonal membrane proteins are first inserted into the dendritic plasma membrane and then reinternalised and trafficked to the axon. (b) Transmembrane proteins, including neurotransmitter receptors, can also be removed from and inserted into the membrane at synapses. Stimulation changes the number of receptors at a synapse which determines the magnitude of synaptic transmissions. On the presynaptic side there are clear vesicles that contain conventional neurotransmitters (e.g. glutamate, GABA) as well as dense‐core vesicles that contain neuropeptides. (c) Many neuropeptides are produced by cleavage of a single large precursor called a prepropeptide in the Golgi and the TGN. Illustrated, a precursor called proopiomelanocortin (POMC) is cleaved up to 8 times to form up to 10 different neuropeptides. (From Castro and Morrison .). (d) As a transmembrane protein is translated, signal sequences in the protein cause it to cross the ER membrane in a certain orientation. Secreted proteins cross the ER membrane a single time. Multiple signal sequences can cause the protein to cross the ER membrane many times (not shown). Adapted from Lodish .
Specialized Cell Structure and Function: Protein Synthesis
N2 - Background: The Ras-related GTPase, Rheb, regulates the growth of animal cells. Genetic and biochemical tests place Rheb upstream of the target of rapamycin (TOR) protein kinase, and downstream of the tuberous sclerosis complex (TSC1/TSC2) and the insulin-signaling pathway. TOR activity is regulated by nutritional cues, suggesting that Rheb might either control, or respond to, nutrient availability. Results: We show that Rheb and TOR do not promote the import of glucose, bulk amino acids, or arginine in Drosophila S2 cells, but that both gene products are important regulators of ribosome biogenesis, protein synthesis, and cell size. S2 cell size, protein synthesis, and glucose import were largely insensitive to manipulations of insulin signaling components, suggesting that cellular energy levels and TOR activity can be maintained through insulin/PI3K-independent mechanisms in S2 cell culture. In vivo in Drosophila larvae, however, we found that insulin signaling can regulate protein synthesis, and thus may affect TOR activity. Conclusion: Rheb-TOR signaling controls S2 cell growth by promoting ribosome production and protein synthesis, but apparently not by direct effects on the import of amino acids or glucose. The effect of insulin signaling upon TOR activity varies according to cellular type and context.
AB - Background: The Ras-related GTPase, Rheb, regulates the growth of animal cells. Genetic and biochemical tests place Rheb upstream of the target of rapamycin (TOR) protein kinase, and downstream of the tuberous sclerosis complex (TSC1/TSC2) and the insulin-signaling pathway. TOR activity is regulated by nutritional cues, suggesting that Rheb might either control, or respond to, nutrient availability. Results: We show that Rheb and TOR do not promote the import of glucose, bulk amino acids, or arginine in Drosophila S2 cells, but that both gene products are important regulators of ribosome biogenesis, protein synthesis, and cell size. S2 cell size, protein synthesis, and glucose import were largely insensitive to manipulations of insulin signaling components, suggesting that cellular energy levels and TOR activity can be maintained through insulin/PI3K-independent mechanisms in S2 cell culture. In vivo in Drosophila larvae, however, we found that insulin signaling can regulate protein synthesis, and thus may affect TOR activity. Conclusion: Rheb-TOR signaling controls S2 cell growth by promoting ribosome production and protein synthesis, but apparently not by direct effects on the import of amino acids or glucose. The effect of insulin signaling upon TOR activity varies according to cellular type and context.
GPCR/G protein Stem Cell Cancer ..
Since E177K exhibited virtually no biological activity, we converted Glu177 to glutamine (E177Q) with the goal of producing an active site mutant that retained some biological activity. This resulted in the expression of pre- and mature RTA proteins that exhibited depurination activity that was approximately 35% of that observed with WT RTA. This agrees with previous studies () demonstrating that the E177Q mutation decreases enzymatic activity at least 170-fold relative to WT RTA in vitro but less than that of the E177K mutation. Interestingly, while ribosome depurination was similar between the pre- and mature forms of E177Q, the degree of protein synthesis inhibition as well as the induction of apoptosis differed. For the pre form of E177Q, protein synthesis was reduced 32% relative to vector control. However, apoptosis was not induced. In contrast, the mature form of E177Q inhibited protein synthesis 60% relative to vector contols which corresponded with full caspase activation and nucleosome accumulation. The reason for the difference in the degree of protein synthesis inhibition when depurination levels were similar is unknown at this time, but may be related to differences in the rate of depurination (). These results indicate that there may be a threshold level of protein synthesis inhibition that correlates with activation of apoptosis in mammalian cells. Interestingly, the difference in the activation of apoptosis did not appear to correlate with differences in activation of JNK or p38, since JNK activation was statistically less for mature E177Q compared to the pre form and there was no difference between the two for p38 activation. Recently it was reported that ricin mediates IL-1β release from bone-marrow derived macrophages through a scaffolding complex termed the NALP3 inflammasome, which facilitates cleavage of pro-IL-1β to active IL-1β by caspase-1. Using inhibitors for proteosome degradation and the JNK and p38 pathways it was concluded that ricin-mediated translation inhibition caused the disappearance of labile proteins that normally suppress inflammasome formation independent of JNK and p38 kinase activation (). Therefore protein synthesis inhibition itself may mediate RTA-induced apoptosis through mechanisms that are independent of stress kinases.
In yeast, we found that S215F and P95L/E145K induced depurination and inhibited protein synthesis similarly to WT preRTA but did not induce nuclear fragmentation and ROS generation, hallmarks of apoptosis. In addition, G212E had low biological activity and did not affect any of these endpoints (). Expression of pre- and mature forms of each of these three mutants reduced depurination levels to 60% of WT RTA control levels in mammalian cells at 19 h after transfection. This lower level of depurination was still sufficient to inhibit protein synthesis by 80 to 90% relative to the vector control. Interestingly, the pre-forms of G212E, S215F, and P95L/E145K did tend to have higher levels of protein synthesis compared to the mature forms or WT preRTA when expressed in mammalian cells, although protein synthesis was still only 15 to 20% of vector control levels. However, they produced full caspase activation, nucleosome accumulation and JNK/p38 signaling. These results indicate that depurination can be reduced by as much as 40% in mammalian cells with minimal effects on protein synthesis inhibition and activation of stress-activated signaling cascades and apoptosis. A substantial reduction in depurination as was observed with pre E177Q may be necessary to prevent protein synthesis inhibition by RTA, possibly due to the high sensitivity of mammalian ribosomes to RTA.
Protein Phosphorylation And Cell Signaling …
Protein Phosphorylation And Cell Signaling Transduction
p53 is a well-studied apoptotic protein crucial for inducing cell-cycle arrest in response to ..
in protein synthesis and oncogenic cell ..
many fundamental cell processes, from protein synthesis to ..
rates of protein synthesis in cell ..
12/04/2007 · Nutrient Signaling Components Controlling Protein Synthesis in ..
Wnt5a protein synthesis via the MAPK signaling ..
To investigate if apoptosis was induced in the transfected cells, activation of caspase 3/7 was investigated in MAC-T cells 19 h after transfection (). Caspase activity was induced similarly by pre- and mature WT RTA (2.7 ± 0.2 and 2.8 ± 0.2-fold, respectively; mean ± SE of 9 experiments) relative to vector controls. The active site mutant E177K elicited negligible caspase activation which corresponded with the lack of ribosome depurination and protein synthesis inhibition. In contrast the pre- and mature forms of E145K, G212E and P95L/E145K as well as mature S215F activated caspase activity to the same degree as their respective WT RTA controls. PreS215F activated caspase 3/7 to a lesser extent than WT preRTA, which corresponded with decreases in depurination and protein synthesis inhibition. The E177Q mutant also showed a difference between the pre- and mature forms in terms of caspase activation, with mature E177Q eliciting a greater increase in caspase 3/7 activity relative to preE177Q. Interestingly, both the pre- and mature forms of P95L activated caspase to a greater extent relative to their WT counterparts.
Cell Communication | Cell Signaling | Protein Kinase
A GFP transfection assay was used to determine if protein synthesis inhibition corresponded with changes in ribosome depurination. Overall, the pattern of ribosome depurination observed with the mutated RTA proteins was reflected in the degree of protein synthesis inhibition (). Fluorescence was almost nondetectable in cells transfected with pre- or mature WT RTA relative to cells transfected with plasmid vector alone. Similar results were obtained with P95L, which depurinated ribosomes similarly to WT RTA based on qRT-PCR results. Pre- and mature E177K did not inhibit protein synthesis which corresponded with the very slight degree of depurination observed. Protein synthesis levels observed with pre- and mature E177Q were intermediate between E177K and the other mutants. Surprisingly, a greater level of inhibition of protein synthesis was observed with mature E177Q (60% inhibition of vector controls) compared to preE177Q (40% inhibition of vector controls) even though they showed similar levels of depurination at 19 h after transfection. Protein synthesis was inhibited significantly less relative to WT RTA for preP95L/E145K and for pre- and mature S215F and G212E, however, this still represented an 80 to 90% inhibition of protein synthesis relative to vector controls. Interestingly, the mature RTA mutants tended to have a greater effect on protein synthesis inhibition than their preRTA counterparts. Specifically, preE177Q, P95L/E145K, S215F and G212E inhibited protein synthesis less than their corresponding mature forms and less than WT preRTA.
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