I observed markedly different weights of a specific organ in my animal model upon treatment. My hypothesis is that this change is largely driven by protein. I would like to examine if the translational activity is changed. How would you adress this? Do you know of any well established markers of protein synthesis activity? I was thinking about eIFs, but this would also relate to ER stress, right?
Any suggestions?
One question! How could you hypothesize that an increase in protein synthesis based on increase in organ weight? Have you done a simple H&E staining of the tissue? There can be many possibilities for increased organ weights such as hyperplasia (e.g. benign tumor), hypertrophy (e.g. enlargement of muscles by exercise) etc. If it is hyperplasia, you can check the proliferation by staining Ki-67 by IHC or BrDU staining; Hypertrophy can be easily detected by measuring the cell size. If your hypothesis is true, then, the tissue should show the latter.
If your treatment increases protein synthesis, most widely used method is incorporation of radio-labelled amino acids. A PubMed search should give you a suitable method/protocol. My suggestion will be first to test your hypothesis in vitro by treating the cells isolated from that organ with your agent and measuring the incorporation of isotope into the protein and then move on to animal studies.
Accumulation of unfolded protein in ER (ER stress) and deficiency of intracellular amino acids (particularly glutamine) causes integrated stress response (ISR). Both amino acid and glucose deprivation, stresses found in solid tumours, activated the upstream eukaryotic initiation factor 2α (eIF2α) kinase GCN2 to up-regulate ATF4 target genes involved in amino acid synthesis and transport. ISR decreases the efficacy of translation of mRNA in general, and then only the transcription and translation of the stress response molecule such as ATF4 and CHOP is attenuated. Up-regulation of ATF4 and CHOP results in the change in regulation of translation via 4E-BP1, translation repressor molecule. The GCN2-eIF2α-ATF4 pathway is critical for maintaining metabolic homeostasis under several stress.
Wnt/mTORC1 crosstalk is deeply associated with ISR-mediated regulation of translation. Wnt3a triggers the activation of mTOR signal, which in turn promotes the protein synthesis. Further, Wnt3a up-regulates GLS (the enzyme which changes glutamine into glutamate, which contributes to mitochondrial TCA cycle) and enhances glutaminolysis. GCN2 is a sensor of intracellular glutamate and GCN2-ATF4 axis is critical for cellular survival by reducing the ER stress and simultaneously making translation more efficient under amino acids deprivation.
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One question! How could you hypothesize that an increase in protein synthesis based on increase in organ weight? Have you done a simple H&E staining of the tissue? There can be many possibilities for increased organ weights such as hyperplasia (e.g. benign tumor), hypertrophy (e.g. enlargement of muscles by exercise) etc. If it is hyperplasia, you can check the proliferation by staining Ki-67 by IHC or BrDU staining; Hypertrophy can be easily detected by measuring the cell size. If your hypothesis is true, then, the tissue should show the latter.
If your treatment increases protein synthesis, most widely used method is incorporation of radio-labelled amino acids. A PubMed search should give you a suitable method/protocol. My suggestion will be first to test your hypothesis in vitro by treating the cells isolated from that organ with your agent and measuring the incorporation of isotope into the protein and then move on to animal studies.
Assessment of blood protein such as albumin transferrin etc they provide information on visceral protein synthesis ?Also lymphocyte blood count is indirectly provide an idea beside measurement of nitro
Have you tried Bio orthogonal chemical labelling followed by mass spectrometry? BONCAT is used to tag and measure newly translated proteins in proteomics. See - http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1480433/
Pay attention!
Generally markers are associated with protein needs of the tissue being studied.
What is the target tissue?
Thank you all for your suggestions and comments. Organ of interest is liver and my 'treatment'= overexpression of an hepatic enzyme by adenovirus.
I am thinking about a very easy, straightforward test such as protein expression of proliferation marker. I will certainly do H&E staining but liver histology does not seem to adress my hypothesis quantitatively.
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