The platform for precise proteomic profiling of targeted cell populations from heterogeneous tissue sections is developed. We demonstrate a seamless and systematic integration of LCM with an automated cap-IA for the handling of a very small-sized dissected tissues section from the kidney, liver and pancreatic Langerhans islet of rats. Our analysis reveals that the lowest LCM section area ≥ 0.125 mm2 with 10 µm thickness can be optimized for the detection of proteins through LCM-cap-IA integration. We detect signals ranging from a highly-abundant protein, β-actin, to a low-abundance protein, LC-3AB, using 0.125 mm2 LCM section from rat kidney, but, so far, a relatively large section is required for good quality of results. This integration is applicable for a highly-sensitive and accurate assessment of microdissected tissue sections to decipher hidden proteomic information of pure targeted cells. To validate this integration, PCK2 protein expression is studied within Langerhans islets of normal and diabetic rats. Our results show significant overexpression of PCK2 in Langerhans islets of rats with long-term diabetes.

• Klíčová slova
• Langerhans islets, PCK2, capillary Western blotting, laser capture microdissection, multiplexing,
• Publikační typ
• časopisecké články MeSH

An increased level of phosphorylation of eukaryotic translation initiation factor 2 subunit-α (eIF2α, encoded by EIF2S1; eIF2α-p) coupled with decreased guanine nucleotide exchange activity of eIF2B is a hallmark of the 'canonical' integrated stress response (c-ISR)1. It is unclear whether impaired eIF2B activity in human diseases including leukodystrophies2, which occurs in the absence of eIF2α-p induction, is synonymous with the c-ISR. Here we describe a mechanism triggered by decreased eIF2B activity, distinct from the c-ISR, which we term the split ISR (s-ISR). The s-ISR is characterized by translational and transcriptional programs that are different from those observed in the c-ISR. Opposite to the c-ISR, the s-ISR requires eIF4E-dependent translation of the upstream open reading frame 1 and subsequent stabilization of ATF4 mRNA. This is followed by altered expression of a subset of metabolic genes (for example, PCK2), resulting in metabolic rewiring required to maintain cellular bioenergetics when eIF2B activity is attenuated. Overall, these data demonstrate a plasticity of the mammalian ISR, whereby the loss of eIF2B activity in the absence of eIF2α-p induction activates the eIF4E-ATF4-PCK2 axis to maintain energy homeostasis.

• MeSH
• energetický metabolismus genetika   MeSH
• eukaryotické iniciační faktory * metabolismus   MeSH
• eukaryotický iniciační faktor 2 metabolismus   MeSH
• eukaryotický iniciační faktor 2B metabolismus   nedostatek   MeSH
• eukaryotický iniciační faktor 4E metabolismus   MeSH
• fosforylace MeSH
• fyziologický stres * genetika   MeSH
• lidé MeSH
• messenger RNA genetika   metabolismus   MeSH
• myši MeSH
• proteosyntéza MeSH
• stabilita RNA MeSH
• transkripční faktor ATF4 genetika   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• ATF4 protein, human MeSH Prohlížeč
• eukaryotické iniciační faktory * MeSH
• eukaryotický iniciační faktor 2 MeSH
• eukaryotický iniciační faktor 2B MeSH
• eukaryotický iniciační faktor 4E MeSH
• messenger RNA MeSH
• transkripční faktor ATF4 MeSH